Bispecific heterodimeric fusion proteins containing IL-15-IL-15Ralpha Fc-fusion proteins and immune checkpoint antibody fragments

ABSTRACT

The present invention is directed to novel bispecific heterodimeric Fc fusion proteins comprising an IL-15/IL-15Rα Fc-fusion protein and a PD-1 antibody fragment-Fc fusion protein.

PRIORITY CLAIM

This application is a continuation of U.S. patent application Ser. No.15/785,393, filed Oct. 16, 2017 which claims priority to U.S. Ser. No.62/408,655, filed on Oct. 14, 2016, U.S. Ser. No. 62/416,087, filed onNov. 1, 2016, U.S. Ser. No. 62/443,465, filed on Jan. 6, 2017, and U.S.Ser. No. 62/477,926, filed on Mar. 28, 2017, which are expressedlyincorporated herein by reference in their entirety, with particularreference to the figures, legends, and claims therein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 17, 2019, isnamed 067461-5202-WO_SL.txt and is 1,801,289 bytes in size.

BACKGROUND OF THE INVENTION

IL-2 and IL-15 function in aiding the proliferation and differentiationof B cells, T cells, and NK cells. IL-2 is also essential for regulatoryT cell (Treg) function and survival. Both cytokines exert their cellsignaling function through binding to a trimeric complex consisting oftwo shared receptors, the common gamma chain (γc; CD132) and IL-2receptor B-chain (IL-2Rβ; CD122), as well as an alpha chain receptorunique to each cytokine: IL-2 receptor alpha (IL-2Rα; CD25) or IL-15receptor alpha (IL-15Rα; CD215). Both cytokines are considered aspotentially valuable therapeutics in oncology and IL-2 has been approvedfor use in patients with metastatic renal-cell carcinoma and malignantmelanoma. Currently there are no approved uses of recombinant IL-15,although several clinical trials are ongoing.

IL-2 presents several challenges as a therapeutic agent. First, itpreferentially activates T cells that express the high affinity receptorcomplex, which depends on CD25 expression. Because Treg cellsconstitutively express CD25, they compete for IL-2 with effector Tcells, whose activation is preferred for oncology treatment. Thisimbalance has led to the concept of high dose IL-2. However, thisapproach creates additional problems because of IL-2-mediated toxicitiessuch as vascular leak syndrome.

IL-2 is secreted primarily by activated T cells, while its receptors arelocated on activated T cells, Tregs, NK cells, and B cells. In contrast,IL-15 is produced on monocytes and dendritic cells and is primarilypresented as a membrane-bound heterodimeric complex with IL-15Rα presenton the same cells. Its effects are realized through trans-presentationof the IL-15/IL-15Rα complex to NK cells and CD8+ T cells expressingIL-2Rβ and the common gamma chain.

As potential drugs, both cytokines suffer from a very fast clearance,with half-lives measured in minutes. In addition, IL-15 by itself isless stable due to its preference for the IL-15Rα-associated complex. Ithas also been shown that recombinantly produced IL-15/IL-15Rαheterodimer can potently activate T cells. Nevertheless, a shorthalf-life hinders favorable dosing.

Checkpoint receptors such as PD-1 (programmed cell death 1) inhibit theactivation, proliferation, and/or effector activities of T cells andother cell types. Guided by the hypothesis that checkpoint receptorssuppress the endogenous T cell response against tumor cells, preclinicaland clinical studies of anti-CTLA4 and anti-PD1 antibodies, includingnivolumab, pembrolizumab, ipilimumab, and tremelimumab, have indeeddemonstrated that immune checkpoint blockade results in impressiveanti-tumor responses, stimulating endogenous T cells to attack tumorcells, leading to long-term cancer remissions in a fraction of patientswith a variety of malignancies. Unfortunately, only a subset of patientsresponds to these therapies, with response rates generally ranging from10 to 30% and sometimes higher for each monotherapy, depending on theindication and other factors. Therapeutic combination of these agents,for example, ipilimumab plus nivolumab, leads to even higher responserates, approaching 60% in some cases. Preclinical studies have shownadditional synergies between anti-PD1 antibodies and/or anti-CTLA4antibodies. While the potential of multiple checkpoint blockade is verypromising, combination therapy with such agents is expected to carry ahigh financial burden. Moreover, autoimmune toxicities of combinationtherapies, for example nivolumab plus ipilimumab, are significantlyelevated compared to monotherapy, causing many patients to halt thetherapy.

Accordingly, the present invention is directed to bispecificheterodimeric fusion proteins that contain an IL-15/IL-15Rα complex-Fcfusion and an antibody fragment that binds to a PD-1 antigen.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides a bispecific heterodimericprotein comprising (a) a fusion protein comprising a first proteindomain, a second protein domain, and a first Fc domain, wherein thefirst protein domain is covalently attached to the N-terminus of thesecond protein domain using a first domain linker, wherein the secondprotein domain is covalently attached to the N-terminus of said first Fcdomain using a second domain linker, and wherein the first proteindomain comprises an IL-15Rα protein and the second protein domaincomprises an IL-15 protein; and (b) an antibody fusion proteincomprising an PD-1 antigen binding domain and a second Fc domain,wherein the PD-1 antigen binding domain is covalently attached to theN-terminus of the second Fc domain, and the PD-1 antigen binding domainis a single chain variable fragment (scFv) or a Fab fragment. The firstand the second Fc domains can have a set of amino acid substitutionsselected from the group consisting ofS267K/L368D/K370S:S267K/LS364K/E357Q; S364K/E357Q:L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411T/E360E/Q362E:D401K;L368D/K370S:S364K/E357L and K370S:S364K/E357Q, according to EUnumbering. In some embodiments, the first and/or the second Fc domainshave an additional set of amino acid substitutions comprisingQ295E/N384D/Q418E/N421D, according to EU numbering. Optionally, thefirst and/or the second Fc domains have an additional set of amino acidsubstitutions consisting of G236R/L328R,E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K,E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G and E233P/L234V/L235A/G236del,according to EU numbering.

In other aspects of the present invention, provided herein is abispecific heterodimeric protein comprising: (a) a fusion proteincomprising a first protein domain and a first Fc domain, wherein thefirst protein domain is covalently attached to the N-terminus of thefirst Fc domain using a domain linker and the first protein domaincomprises an IL-15Rα protein; (b) a second protein domain noncovalentlyattached to the first protein domain, the second protein domaincomprises an IL-15 protein; and (c) an antibody fusion proteincomprising an PD-1 antigen binding domain and a second Fc domain,wherein the PD-1 antigen binding domain is covalently attached to theN-terminus of the second Fc domain and said PD-1 antigen binding domainis a single chain variable fragment (scFv) or a Fab fragment. The firstand the second Fc domains can have a set of amino acid substitutionsselected from the group consisting ofS267K/L368D/K370S:S267K/LS364K/E357Q; S364K/E357Q:L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411T/E360E/Q362E:D401K;L368D/K370S:S364K/E357L and K370S:S364K/E357Q, according to EUnumbering. In some embodiments, the first and/or the second Fc domainshave an additional set of amino acid substitutions comprisingQ295E/N384D/Q418E/N421D, according to EU numbering. In other instances,the first and/or the second Fc domains have an additional set of aminoacid substitutions consisting of G236R/L328R,E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K,E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G and E233P/L234V/L235A/G236del,according to EU numbering.

In some aspects of the present invention, provided herein is abispecific heterodimeric protein comprising: (a) a first antibody fusionprotein comprising a first PD-1 antigen binding domain and a first Fcdomain, wherein the first PD-1 antigen binding domain is covalentlyattached to the N-terminus of the first Fc domain via a first domainlinker, and the first PD-1 antigen binding domain is a single chainvariable fragment (scFv) or a Fab fragment; (b) a second antibody fusionprotein comprising a second PD-1 antigen binding domain, a second Fcdomain, and a first protein domain, wherein the second PD-1 antigenbinding domain is covalently attached to the N-terminus of the second Fcdomain via a second domain linker, the first protein domain iscovalently attached to the C-terminus of the second Fc domainvia a thirddomain linker, the second PD-1 antigen binding domain is a single chainvariable fragment (scFv) or a Fab fragment, and the first protein domaincomprises an IL-15Rα protein; and (c) a second protein domainnoncovalently attached to the first protein domain of the secondantibody fusion protein and comprising an IL-15 protein. In someembodiments, the first and the second Fc domains can have a set of aminoacid substitutions selected from the group consisting ofS267K/L368D/K370S:S267K/LS364K/E357Q; S364K/E357Q:L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411T/E360E/Q362E:D401K;L368D/K370S:S364K/E357L and K370S:S364K/E357Q, according to EUnumbering. In some embodiments, the first and/or the second Fc domainshave an additional set of amino acid substitutions comprisingQ295E/N384D/Q418E/N421D, according to EU numbering. In other instances,the first and/or the second Fc domains have an additional set of aminoacid substitutions consisting of G236R/L328R,E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K,E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G and E233P/L234V/L235A/G236del,according to EU numbering.

In some aspects, the IL15 protein of the bispecific heterodimericprotein described herein has one or more amino acid substitutionsselected from the group consisting of N1D, N4D, D8N, D30N, D61N, E64Q,N65D, and Q108E.

In other aspects, provided herein is a bispecific heterodimeric proteinselected from the group consisting of XENP25850, XENP25937, XENP21480,XENP22022, XENP22112, XENP22641, XENP22642, and XENP22644.

Nucleic acids, expression vectors and host cells are all provided aswell, in addition to methods of making these proteins and treatingpatients with them.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structure of IL-15 in complex with its receptorsIL-15Rα (CD215), IL-15Rß (CD122), and the common gamma chain (CD132).

FIG. 2A-2B depicts the sequences for IL-15 and its receptors.

FIG. 3A-3E depicts useful pairs of Fc heterodimerization variant sets(including skew and pI variants). There are variants for which there areno corresponding “monomer 2” variants; these are pI variants which canbe used alone on either monomer. In addition, reference is made to FIG.88 .

FIG. 4 depict a list of isosteric variant antibody constant regions andtheir respective substitutions. pI_(−) indicates lower pI variants,while pI_(+) indicates higher pI variants. These can be optionally andindependently combined with other heterodimerization variants of theinventions (and other variant types as well, as outlined herein.)

FIG. 5 depict useful ablation variants that ablate FcγR binding(sometimes referred to as “knock outs” or “KO” variants). Generally,ablation variants are found on both monomers, although in some casesthey may be on only one monomer.

FIG. 6A-6E shows a particularly useful embodiments of “non-cytokine”components of the IL-15/Ra-Fc fusion proteins of the invention, as filedconcurrently with the present case on 16 Oct. 2017 entitled “IL15/IL15RαHeterodimeric Fc-fusion Proteins” and U.S. Ser. No. 62/408,655, filed onOct. 14, 2016, U.S. Ser. No. 62/443,465, filed on Jan. 6, 2017, and U.S.Ser. No. 62/477,926, filed on Mar. 28, 2017, hereby incorporated byreference in their entirety and in particular for the sequences outlinedtherein.

FIG. 7A-7F shows particularly useful embodiments of“non-cytokine”/“non-Fv” components of the IL-15/Rα×anti-PD-1bifunctional proteins of the invention. For each, the inclusion of the428L/434S FcRn half life extension variants can be added.

FIG. 8 depicts a number of exemplary variable length linkers for use inIL-15/Ra-Fc fusion proteins. In some embodiments, these linkers find uselinking the C-terminus of IL-15 and/or IL-15Rα(sushi) to the N-terminusof the Fc region. In some embodiments, these linkers find use fusingIL-15 to the IL-15Rα(sushi).

FIG. 9A-9C depict a number of charged scFv linkers that find use inincreasing or decreasing the pI of heterodimeric fusion proteins thatutilize one or more scFv as a component. The (+H) positive linker findsparticular use herein. A single prior art scFv linker with single chargeis referenced as “Whitlow”, from Whitlow et al., Protein Engineering6(8):989-995 (1993). It should be noted that this linker was used forreducing aggregation and enhancing proteolytic stability in scFvs.

FIG. 10A-10D shows the sequences of several useful IL-15/Rα-Fc formatbackbones based on human IgG1, without the cytokine sequences (e.g. the11-15 and/or IL-15Rα(sushi)). It is important to note that thesebackbones can also find use in certain embodiments of IL-15/Rα×anti-PD-1bifunctional proteins. Backbone 1 is based on human IgG1 (356E/358Mallotype), and includes C220S on both chains, theS364K/E357Q:L368D/K370S skew variants, the Q295E/N384D/Q418E/N421D pIvariants on the chain with L368D/K370S skew variants and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains.Backbone 2 is based on human IgG1 (356E/358M allotype), and includesC220S on both chain, the S364K:L368D/K370S skew variants, theQ295E/N384D/Q418E/N421D pI variants on the chain with L368D/K370S skewvariants and the E233P/L234V/L235A/G236del/S267K ablation variants onboth chains. Backbone 3 is based on human IgG1 (356E/358M allotype), andincludes C220S on both chain, the S364K:L368E/K370S skew variants, theQ295E/N384D/Q418E/N421D pI variants on the chain with L368E/K370S skewvariants and the E233P/L234V/L235A/G236del/S267K ablation variants onboth chains. Backbone 4 is based on human IgG1 (356E/358M allotype), andincludes C220S on both chain, the D401K: K360E/Q362E/T411E skewvariants, the Q295E/N384D/Q418E/N421D pI variants on the chain withK360E/Q362E/T411E skew variants and the E233P/L234V/L235A/G236del/S267Kablation variants on both chains. Backbone 5 is based on human IgG1(356D/358L allotype), and includes C220S on both chain, theS364K/E357Q:L368D/K370S skew variants, the Q295E/N384D/Q418E/N421D pIvariants on the chain with L368D/K370S skew variants and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains.Backbone 6 is based on human IgG1 (356E/358M allotype), and includesC220S on both chain, the S364K/E357Q:L368D/K370S skew variants,Q295E/N384D/Q418E/N421D pI variants on the chain with L368D/K370S skewvariants and the E233P/L234V/L235A/G236del/S267K ablation variants onboth chains, as well as an N297A variant on both chains. Backbone 7 isidentical to 6 except the mutation is N297S. Alternative formats forbackbones 6 and 7 can exclude the ablation variantsE233P/L234V/L235A/G236del/S267K in both chains. Backbone 8 is based onhuman IgG4, and includes the S364K/E357Q:L368D/K370S skew variants, theQ295E/N384D/Q418E/N421D pI variants on the chain with L368D/K370S skewvariants, as well as a S228P (EU numbering, this is S241P in Kabat)variant on both chains that ablates Fab arm exchange as is known in theart. Backbone 9 is based on human IgG2, and includes theS364K/E357Q:L368D/K370S skew variants, the Q295E/N384D/Q418E/N421D pIvariants on the chain with L368D/K370S skew variants. Backbone 10 isbased on human IgG2, and includes the S364K/E357Q:L368D/K370S skewvariants, the Q295E/N384D/Q418E/N421D pI variants on the chain withL368D/K370S skew variants as well as a S267K variant on both chains.Backbone 11 is identical to backbone 1, except it includes M428L/N434SXtend mutations. Backbone 12 is based on human IgG1 (356E/358Mallotype), and includes C220S on both identical chain, theE233P/L234V/L235A/G236del/S267K ablation variants on both identicalchains. Backbone 13 is based on human IgG1 (356E/358M allotype), andincludes C220S on both chain, the S364K/E357Q:L368D/K370S skew variants,the P217R/P229R/N276K pI variants on the chain with S364K/E357Q skewvariants and the E233P/L234V/L235A/G236del/S267K ablation variants onboth chains.

As will be appreciated by those in the art and outlined below, thesesequences can be used with any IL-15 and IL-15Rα(sushi) pairs outlinedherein, including but not limited to IL-15/Rα-heteroFc, ncIL-15/Rα, andscIL-15/Rα, as schematically depicted in FIGS. 64A-64K. Additionally,any IL-15 and/or IL-15Rα(sushi) variants can be incorporated into theseFIG. 10 backbones in any combination.

Included within each of these backbones are sequences that are 90, 95,98 and 99% identical (as defined herein) to the recited sequences,and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional aminoacid substitutions (as compared to the “parent” of the Figure, which, aswill be appreciated by those in the art, already contain a number ofamino acid modifications as compared to the parental human IgG1 (or IgG2or IgG4, depending on the backbone). That is, the recited backbones maycontain additional amino acid modifications (generally amino acidsubstitutions) in addition to the skew, pI and ablation variantscontained within the backbones of this figure.

FIG. 11 shows the sequences of several useful IL-15/Rα×anti-PD-1bifunctional format backbones based on human IgG1, without the cytokinesequences (e.g. the 11-15 and/or IL-15Rα(sushi)) or VH, and furtherexcluding light chain backbones which are depicted in FIGS. 10A-10D.Backbone 1 is based on human IgG (356E/358M allotype), and includes theS364K/E357Q:L368D/K370S skew variants, C220S and theQ295E/N384D/Q418E/N421D pI variants on the chain with L368D/K370S skewvariants and the E233P/L234V/L235A/G236del/S267K ablation variants onboth chains. Backbone 2 is based on human IgG (356E/358M allotype), andincludes the S364K/E357Q:L368D/K370S skew variants, theN208D/Q295E/N384D/Q418E/N421D pI variants on the chain with L368D/K370Sskew variants, C220S in the chain with S364K/E357Q variants, and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains.Backbone 3 is based on human IgG (356E/358M allotype), and includes theS364K/E357Q:L368D/K370S skew variants, the N208D/Q295E/N384D/Q418E/N421DpI variants on the chains with L368D/K370S skew variants, the Q196K/I199T/P217R/P228R/N276K pI variants on the chains with S364K/E357Qvariants, and the E233P/L234V/L235A/G236del/S267K ablation variants onboth chains.

In certain embodiments, these sequences can be of the 356D/358Lallotype. In other embodiments, these sequences can include either theN297A or N297S substitutions. In some other embodiments, these sequencescan include the M428L/N434S Xtend mutations. In yet other embodiments,these sequences can instead be based on human IgG4, and include a S228P(EU numbering, this is S241P in Kabat) variant on both chains thatablates Fab arm exchange as is known in the art. In yet furtherembodiments, these sequences can instead be based on human IgG2.Further, these sequences may instead utilize the other skew variants, pIvariants, and ablation variants depicted in FIG. 3 .

As will be appreciated by those in the art and outlined below, thesesequences can be used with any IL-15 and IL-15Rα(sushi) pairs outlinedherein, including but not limited to scIL-15/Rα, ncIL-15/Rα, anddsIL-15Rα, as schematically depicted in FIGS. 64A-64K. Further as willbe appreciated by those in the art and outlined below, any IL-15 and/orIL-15Rα(sushi) variants can be incorporated in these backbones.Furthermore as will be appreciated by those in the art and outlinedbelow, these sequences can be used with any VH and VL pairs outlinedherein, including either a scFv or a Fab.

Included within each of these backbones are sequences that are 90, 95,98 and 99% identical (as defined herein) to the recited sequences,and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional aminoacid substitutions (as compared to the “parent” of the Figure, which, aswill be appreciated by those in the art, already contain a number ofamino acid modifications as compared to the parental human IgG1 (or IgG2or IgG4, depending on the backbone). That is, the recited backbones maycontain additional amino acid modifications (generally amino acidsubstitutions) in addition to the skew, pI and ablation variantscontained within the backbones of this figure.

FIG. 12 depicts the “non-Fv” backbone of light chains (i.e. constantlight chain) which find use in IL-15/Rα×anti-PD-1 bifunctional proteinsof the invention.

FIG. 13A-13E depicts the sequences for a select number of anti-PD-1antibodies. It is important to note that these sequences were generatedbased on human IgG1, with an ablation variant(E233P/L234V/L235A/G236del/S267K, “IgG1_PVA_S267k”). The CDRs areunderlined. As noted herein and is true for every sequence hereincontaining CDRs, the exact identification of the CDR locations may beslightly different depending on the numbering used as is shown in Table1, and thus included herein are not only the CDRs that are underlinedbut also CDRs included within the VH and VL domains using othernumbering systems.

FIG. 14A-14F depict a select number of PD-1 ABDs, with additionalanti-PD-1 ABDs being listed as SEQ ID Nos: XXX. The CDRs are underlined,the scFv linker is double underlines (in the sequences, the scFv linkeris a positively charged scFv (GKPGS)₄ linker (SEQ ID NO: 5), although aswill be appreciated by those in the art, this linker can be replaced byother linkers, including uncharged or negatively charged linkers, someof which are depicted in FIGS. 9A-9C, and the slashes indicate theborder(s) of the variable domains> In addition, the naming conventionillustrates the orientation of the scFv from N to C-terminus; some ofthe sequences in this Figure are oriented as V_(H)-scFv linker-V_(L)(from N- to C-terminus), while some are oriented as V_(L)-scFvlinker-V_(H) (from N- to C-terminus), although as will be appreciated bythose in the art, these sequences may also be used in the oppositionorientation from their depiction herein. As noted herein and is true forevery sequence herein containing CDRs, the exact identification of theCDR locations may be slightly different depending on the numbering usedas is shown in Table 1, and thus included herein are not only the CDRsthat are underlined but also CDRs included within the VH and V_(L)domains using other numbering systems. Furthermore, as for all thesequences in the Figures, these V_(H) and V_(L) sequences can be usedeither in a scFv format or in a Fab format.

FIG. 15 depicts the sequences for XENP21575, a chimeric anti-PD-1antibody based on the variable regions of hybridoma clone 1C11 and humanIgG1 with E233P/L234V/L235A/G236del/S267K substitutions in the heavychain. The CDRs are in bold, and the slashes indicate the borders of thevariable domains. As note herein and is true for every sequence hereincontaining CDRs, the exact identification of the CDR locations may beslightly different depending on numbering used as is shown in Table 1,and thus included herein are not only the CDRs that are underlined butalso CDRs included within the VH and VL domains using other numberingsystems.

FIG. 16 depicts blocking of PD-1/PD-L1 interaction on PD-1 transfectedHEK293T cells by anti-PD-1 clone 1C11.

FIG. 17 depicts the binding of anti-PD-1 clone 1C11 to SEB-stimulated Tcells.

FIG. 18A-18B depicts cytokine release assays (A: IL-2; B: IFNγ) afterSEB stimulation of human PBMCs and treatment with anti-PD-1 clone 1C11.

FIG. 19 depicts the sequences for an illustrative humanized variant ofanti-PD-1 clone 1C11 in bivalent antibody (XENP22553) in the human IgG1format with E233P/L234V/L235A/G236del/S267K substitutions in the heavychain. The CDRs are in bold, and the slashes indicate the borders of thevariable domains. As note herein and is true for every sequence hereincontaining CDRs, the exact identification of the CDR locations may beslightly different depending on numbering used as is shown in Table 1,and thus included herein are not only the CDRs that are underlined butalso CDRs included within the VH and VL domains using other numberingsystems. Sequences for additional humanized variants of anti-PD-1 clone1C11 are depicted as SEQ ID NOs: 832-931 and 942-951 (include XENPs22543, 22544, 22545, 22546, 22547, 22548, 22549, 22550, 22551, 22552,and 22554). As will be appreciated by those in the art, the VH and VLdomains can be formatted as Fab or scFvs for use in theIL-15/Rα×anti-PD-1 bifunctional proteins of the invention.

FIG. 20 depicts the affinity of XENP22553 for PD-1 as determined byOctet (as well as the associated sensorgram).

FIG. 21 A-21G depict several formats for the IL-15/Rα-Fc fusion proteinsof the present invention. IL-15Rα Heterodimeric Fc fusion or“IL-15/Rα-heteroFc” (FIG. 21A) comprises IL-15 recombinantly fused toone side of a heterodimeric Fc and IL-15Rα(sushi) recombinantly fused tothe other side of a heterodimeric Fc. The IL-15 and IL-15Rα(sushi) mayhave a variable length Gly-Ser linker between the C-terminus and theN-terminus of the Fc region. Single-chain IL-15/Rα-Fc fusion or“scIL-15/Rα-Fc” (FIG. 21B) comprises IL-15Rα(sushi) fused to IL-15 by avariable length linker (termed a “single-chain” IL-15/IL-15Rα(sushi)complex or “scIL-15/Rα”) which is then fused to the N-terminus of aheterodimeric Fc-region, with the other side of the molecule being“Fc-only” or “empty Fc”. Non-covalent IL-15/Rα-Fc or “ncIL-15/Rα-Fc”(FIG. 21C) comprises IL-15Rα(sushi) fused to a heterodimeric Fc region,while IL-15 is transfected separatedly so that a non-covalent IL-15/Rαcomplex is formed, with the other side of the molecule being “Fc-only”or “empty Fc”. Bivalent non-covalent IL-15/Rα-Fc fusion or “bivalentncIL-15/Rα-Fc” (FIG. 21D) comprises IL-15Rα(sushi) fused to theN-terminus of a homodimeric Fc region, while IL-15 is transfectedseparately so that a non-covalent IL-15/Rα complex is formed. Bivalentsingle-chain IL-15/Rα-Fc fusion or “bivalent scIL-15/Rα-Fc” (FIG. 21E)comprises IL-15 fused to IL-15Rα(sushi) by a variable length linker(termed a “single-chain” IL-15/IL-15Rα(sushi) complex or “scIL-15/Rα”)which is then fused to the N-terminus of a homodimeric Fc-region.Fc-non-covalent IL-15/Rα fusion or “Fc-ncIL-15/Rα” (FIG. 21F) comprisesIL-15Rα(sushi) fused to the C-terminus of a heterodimeric Fc region,while IL-15 is transfected separately so that a non-covalent IL-15/Rαcomplex is formed, with the other side of the molecule being “Fc-only”or “empty Fc”. Fc-single-chain IL-15/Rα fusion or “Fc-scIL-15/Rα” (FIG.21G) comprises IL-15 fused to IL-15Rα(sushi) by a variable length linker(termed a “single-chain” IL-15/IL-15Rα(sushi) complex or “scIL-15/Rα”)which is then fused to the C-terminus of a heterodimeric Fc region, withthe other side of the molecule being “Fc-only” or “empty Fc”.

FIG. 22 depicts sequences of XENP20818 and XENP21475, illustrativeIL-15/Rα-Fc fusion proteins of the “IL-15/Rα-heteroFc” format, withadditional sequences being listed as XENPs 20819, 21471, 21472, 21473,21474, 21476, and 21477 in the Figures. IL-15 and IL-15Rα(sushi) areunderlined, linkers are double underlined (although as will beappreciated by those in the art, the linkers can be replaced by otherlinkers, some of which are depicted in FIG. 7 ), and slashes (/)indicate the border(s) between IL-15, IL-15Rα, linkers, and Fc regions.

FIG. 23 depicts sequences of XENP21478, an illustrative IL-15/Rα-Fcfusion protein of the “scIL-15/Rα-Fc” format, with additional sequencesbeing listed as SEQ ID NOs: XXX-YYY (include XENPs 21993, 21994, 21995,23174, 23175, 24477, and 24480). IL-15 and IL-15Rα(sushi) areunderlined, linkers are double underlined (although as will beappreciated by those in the art, the linkers can be replaced by otherlinkers, some of which are depicted in FIG. 8 ), and slashes (/)indicate the border(s) between IL-15, IL-15Rα, linkers, and Fc regions.

FIG. 24 depicts sequences of XENP21479, XENP22366 and XENP24348,illustrative IL-15/Rα-Fc fusion proteins of the “ncIL-15/Rα-Fc” format.IL-15 and IL-15Rα(sushi) are underlined, linkers are double underlined(although as will be appreciated by those in the art, the linkers can bereplaced by other linkers, some of which are depicted in FIG. 8 ), andslashes (/) indicate the border(s) between IL-15, IL-15Rα, linkers, andFc regions.

FIG. 25 depicts sequences of XENP21978, an illustrative IL-15/Rα-Fcfusion protein of the “bivalent ncIL-15/Rα-Fc” format, with additionalsequences being listed as SEQ ID NOs: XXX-YYY (include XENP21979). IL-15and IL-15Rα(sushi) are underlined, linkers are double underlined(although as will be appreciated by those in the art, the linkers can bereplaced by other linkers, some of which are depicted in FIG. 7 ), andslashes (/) indicate the border(s) between IL-15, IL-15Rα, linkers, andFc regions.

FIG. 26 depicts sequences of an illustrative IL-15/Rα-Fc fusion proteinof the “bivalent scIL-15/Rα-Fc” format. IL-15 and IL-15Rα(sushi) areunderlined, linkers are double underlined (although as will beappreciated by those in the art, the linkers can be replaced by otherlinkers, some of which are depicted in FIG. 7 ), and slashes (/)indicate the border(s) between IL-15, IL-15Rα, linkers, and Fc regions.

FIG. 27 depicts sequences of XENP22637, an illustrative IL-15/Rα-Fcfusion protein of the “Fc-ncIL-15/Rα” format, with additional sequencesbeing listed as SEQ ID NOs: XXX-YYY (include XENP22638). IL-15 andIL-15Rα(sushi) are underlined, linkers are double underlined (althoughas will be appreciated by those in the art, the linkers can be replacedby other linkers, some of which are depicted in FIG. 8 ), and slashes(/) indicate the border(s) between IL-15, IL-15Rα, linkers, and Fcregions.

FIG. 28 depicts sequences of an illustrative IL-15/Rα-Fc fusion proteinof the “Fc-scIL-15/Rα” format. IL-15 and IL-15Rα(sushi) are underlined,linkers are double underlined (although as will be appreciated by thosein the art, the linkers can be replaced by other linkers, some of whichare depicted in FIG. 8 ), and slashes (/) indicate the border(s) betweenIL-15, IL-15Rα, linkers, and Fc regions.

FIG. 29A-29C depicts the induction of A) NK (CD56⁺/CD16⁺) cells, B) CD4⁺T cells, and C) CD8⁺ T cells proliferation by illustrative IL-15/Rα-Fcfusion proteins of Format A with different linker lengths based on Ki67expression as measured by FACS.

FIG. 30A-30C depicts the induction of A) NK (CD56/CD16⁺) cells, B) CD4⁺T cells, and C) CD8⁺ T cells proliferation by illustrative IL-15/Rα-Fcfusion proteins of scIL-15/Rα-Fc format (XENP21478) and ncIL-15/Rα-Fcformat (XENP21479) based on Ki67 expression as measured by FACS.

FIG. 31 depicts a structural model of the IL-15/Rα heterodimer showinglocations of engineered disulfide bond pairs.

FIG. 32 depicts sequences for illustrative IL-15Rα(sushi) variantsengineered with additional residues at the C-terminus to serve as ascaffold for engineering cysteine residues.

FIG. 33 depicts sequences for illustrative IL-15 variants engineeredwith cysteines in order to form covalent disulfide bonds withIL-15Rα(sushi) variants engineered with cysteines.

FIG. 34 depicts sequences for illustrative IL-15Rα(sushi) variantsengineered with cysteines in order to form covalent disulfide bonds withIL-15 variants engineered with cysteines.

FIG. 35A-35D depicts additional formats for the IL-15/Rα-Fc fusionproteins of the present invention with engineered disulfide bonds.Disulfide-bonded IL-15/Rα heterodimeric Fc fusion or“dsIL-15/Rα-heteroFc” (FIG. 35A) is the same as “IL-15/Rα-heteroFc”, butwherein IL-15Rα(sushi) and IL-15 are further covalently linked as aresult of engineered cysteines. Disulfide-bonded IL-15/Rα Fc fusion or“dsIL-15/Rα-Fc” (FIG. 35B) is the same as “ncIL-15/Rα-Fc”, but whereinIL-15Rα(sushi) and IL-15 are further covalently linked as a result ofengineered cysteines. Bivalent disulfide-bonded IL-15/Rα-Fc or “bivalentdsIL-15/Rα-Fc” (FIG. 35C) is the same as “bivalent ncIL-15/Rα-Fc”, butwherein IL-15Rα(sushi) and IL-15 are further covalently linked as aresult of engineered cysteines. Fc-disulfide-bonded IL-15/Rα fusion or“Fc-dsIL-15/Rα” (FIG. 35D) is the same as “Fc-ncIL-15/Rα”, but whereinIL-15Rα(sushi) and IL-15 are further covalently linked as a result ofengineered cysteines.

FIG. 36A-36B depicts sequences of XENP22013, XENP22014, XENP22015, andXENP22017, illustrative IL-15/Rα-Fc fusion protein of the“dsIL-15/Rα-heteroFc” format. IL-15 and IL-15Rα(sushi) are underlined,linkers are double underlined (although as will be appreciated by thosein the art, the linkers can be replaced by other linkers, some of whichare depicted in FIG. 8 ), and slashes (/) indicate the border(s) betweenIL-15, IL-15Rα, linkers, and Fc regions.

FIG. 37A-37B depicts sequences of XENP22357, XENP22358, XENP22359,XENP22684, and XENP22361, illustrative IL-15/Rα-Fc fusion proteins ofthe “dsIL-15/Rα-Fc” format. Additional sequences are depicted XENPs22360, 22362, 22363, 22364, 22365, and 22366). IL-15 and IL-15Rα(sushi)are underlined, linkers are double underlined (although as will beappreciated by those in the art, the linkers can be replaced by otherlinkers, some of which are depicted in FIG. 8 ), and slashes (/)indicate the border(s) between IL-15, IL-15Rα, linkers, and Fc regions.

FIG. 38 depicts sequences of XENP22634, XENP22635, and XENP22636,illustrative IL-15/Rα-Fc fusion proteins of the “bivalent dsIL-15/Rα-Fc”format. Additional sequences are depicted as SEQ ID NOs: XXX-YYY(include XENP22687). IL-15 and IL-15Rα(sushi) are underlined, linkersare double underlined (although as will be appreciated by those in theart, the linkers can be replaced by other linkers, some of which aredepicted in FIG. 8 ), and slashes (/) indicate the border(s) betweenIL-15, IL-15Rα, linkers, and Fc regions.

FIG. 39 depicts sequences of XENP22639 and XENP22640, illustrativeIL-15/Rα-Fc fusion proteins of the “Fc-dsIL-15/Rα” format. IL-15 andIL-15Rα(sushi) are underlined, linkers are double underlined (althoughas will be appreciated by those in the art, the linkers can be replacedby other linkers, some of which are depicted in FIG. 8 ), and slashes(/) indicate the border(s) between IL-15, IL-15Rα, linkers, and Fcregions.

FIG. 40 depicts the purity and homogeneity of illustrative IL-15/Rα-Fcfusion proteins with and without engineered disulfide bonds asdetermined by CEF.

FIG. 41A-41C depicts the induction of A) NK (CD56+/CD16+) cell, B) CD8⁺T cell, and C) CD4⁺ T cell proliferation by illustrative IL-15/Rα-Fcfusion proteins with and without engineered disulfide bonds based onKi67 expression as measured by FACS.

FIG. 42 depicts the structure of IL-15 complexed with IL-15Rα, IL-2RB,and common gamma chain. Locations of substitutions designed to reducepotency are shown.

FIG. 43A-43C depicts sequences for illustrative IL-15 variantsengineered for reduced potency. Included within each of these variantIL-15 sequences are sequences that are 90, 95, 98 and 99% identical (asdefined herein) to the recited sequences, and/or contain from 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions. In anon-limiting example, the recited sequences may contain additional aminoacid modifications such as those contributing to formation of covalentdisulfide bonds as described in Example 3B.

FIG. 44A-44D depicts sequences of XENP22821, XENP22822, XENP23554,XENP23557, XENP23561, XENP24018, XENP24019, XENP24045, XENP24051, andXENP24052, illustrative IL-15/Rα-Fc fusion proteins of the“IL-15/Rα-heteroFc” format engineered for reduced potency. Additionalsequences are depicted as SEQ ID NOs: XXX-YYY (include XENPs 22815,22816, 22817, 22818, 22819, 22820, 22823, 22824, 22825, 22826, 22827,22828, 22829, 22830, 22831, 22832, 22833, 22834, 23555, 23559, 23560,24017, 24020, 24043, and 24048). IL-15 and IL-15Rα(sushi) areunderlined, linkers are double underlined (although as will beappreciated by those in the art, the linkers can be replaced by otherlinkers, some of which are depicted in FIG. 8 ), and slashes (/)indicate the border(s) between IL-15, IL-15Rα, linkers, and Fc regions.

FIG. 45A-45C depicts sequences of XENP24015, XENP24050, XENP24475,XENP24476, XENP24478, XENP24479, and XENP24481, illustrative IL-15/Rα-Fcfusion proteins of the “scIL-15/Rα-Fc” format engineered for reducedpotency. Additional sequences are depicted as SEQ ID NOs: XXX-YYY(include XENPs 24013, 24014, and 24016). IL-15 and IL-15Rα(sushi) areunderlined, linkers are double underlined (although as will beappreciated by those in the art, the linkers can be replaced by otherlinkers, some of which are depicted in FIG. 8 , and slashes (/) indicatethe border(s) between IL-15, IL-15Rα, linkers, and Fc regions.

FIG. 46A-46B depicts sequences of XENP24349, XENP24890, and XENP25138,illustrative IL-15/Rα-Fc fusion proteins of the “ncIL-15/Rα-Fc” formatengineered for reduced potency. IL-15 and IL-15Rα(sushi) are underlined,linkers are double underlined (although as will be appreciated by thosein the art, the linkers can be replaced by other linkers, some of whichare depicted in FIG. 8 ), and slashes (/) indicate the border(s) betweenIL-15, IL-15Rα, linkers, and Fc regions.

FIG. 47 depicts sequences of XENP22801 and XENP22802, illustrativencIL-15/Rα heterodimers engineered for reduced potency. Additionalsequences are depicted as SEQ ID NOs: XXX-YYY (XENPs 22791, 22792,22793, 22794, 22795, 22796, 22803, 22804, 22805, 22806, 22807, 22808,22809, 22810, 22811, 22812, 22813, and 22814). It is important to notethat these sequences were generated using polyhistidine (His×6 or HHHHHH(SEQ ID NO: 6)) C-terminal tags at the C-terminus of IL-15Rα(sushi).

FIG. 48 depicts sequences of XENP24342, an illustrative IL-15/Rα-Fcfusion protein of the “bivalent ncIL-15/Rα-Fc” format engineered forreduced potency. IL-15 and IL-15Rα(sushi) are underlined, linkers aredouble underlined (although as will be appreciated by those in the art,the linkers can be replaced by other linkers, some of which are depictedin FIG. 8 ), and slashes (/) indicate the border(s) between IL-15,IL-15Rα, linkers, and Fc regions.

FIG. 49 depicts sequences of XENP23472 and XENP23473, illustrativeIL-15/Rα-Fc fusion proteins of the “dsIL-15/Rα-Fc” format engineered forreduced potency. IL-15 and IL-15Rα(sushi) are underlined, linkers aredouble underlined (although as will be appreciated by those in the art,the linkers can be replaced by other linkers, some of which are depictedin FIG. 8 ), and slashes (/) indicate the border(s) between IL-15,IL-15Rα, linkers, and Fc regions.

FIG. 50A-50C depicts the induction of A) NK cell, B) CD8+ (CD45RA−) Tcell, and C) CD4+ (CD45RA−) T cell proliferation by variant IL-15/Rα-Fcfusion proteins based on Ki67 expression as measured by FACS.

FIG. 51 depicts EC50 for induction of NK and CD8⁺ T cells proliferationby variant IL-15/Rα-Fc fusion proteins, and fold reduction in EC50relative to XENP20818.

FIG. 52A-52C depicts the gating of lymphocytes and subpopulations forthe experiments depicted in FIG. 51 . FIG. 52A shows the gatedlymphocyte population.

FIG. 52B shows the CD3-negative and CD3-positive subpopulations. FIG.52C shows the CD16=negative and CD16-positive subpopulations of theCD3-negative cells.

FIG. 53A-53C depicts the gating of CD3⁺ lymphocyte subpopulations forthe experiments depicted in FIG. 51 . FIG. 53A shows the CD4⁺, CD8⁺ andγδ T cell subpopulations of the CD3⁺ T cells. FIG. 53B shows theCD45RA(−) and CD45RA(+) subpopulations of the CD4⁺ T cells. FIG. 53Cshows the CD45RA(−) and CD45RA(+) subpopulation s of the CD8⁺ T cells.

FIG. 54A-54B depicts CD69 and CD25 expression before (FIG. 54A) andafter (FIG. 54B) incubation of human PBMCs with XENP22821

FIG. 55A-55D depict cell proliferation in human PBMCs incubated for fourdays with the indicated variant IL-15/Rα-Fc fusion proteins. FIGS.55A-55D show the percentage of proliferating NK cells (CD3−CD16+) (FIG.55A), CD8⁺ T cells (CD3+CD8+CD45RA−) (FIG. 55B) and CD4⁺ T cells(CD3+CD4+CD45RA−) (FIG. 55C). FIG. 55D shows the fold change in EC50 ofvarious IL15/IL15Rα Fc heterodimers relative to control (XENP20818).

FIG. 56A-56D depict cell proliferation in human PBMCs incubated forthree days with the indicated variant IL-15/Rα-Fc fusion proteins. FIGS.56A-56D show the percentage of proliferating CD8⁺ (CD45RA−) T cells(FIG. 56A), CD4⁺ (CD45RA−) T cells (FIG. 56B), γδ T cells (FIG. 56C),and NK cells (FIG. 56D).

FIG. 57A-57C depicts the percentage of Ki67 expression on (A) CD8⁺ Tcells, (B) CD4⁺ T cells, and (C) NK cells following treatment withadditional IL-15/Rα variants.

FIG. 58A-58E depicts the percentage of Ki67 expression on (A) CD8⁺(CD45RA−) T cells, (B) CD4⁺ (CD45RA−) T cells, (C) γδ T cells, (D) NK(CD16+CD8α−) cells, and (E) NK (CD56+CD8α−) cells following treatmentwith IL-15/Rα variants.

FIG. 59A-59E depicts the percentage of Ki67 expression on (A) CD8⁺(CD45RA−) T cells, (B) CD4⁺ (CD45RA−) T cells, (C) γδ T cells, (D) NK(CD16+CD8α−) cells, and (E) NK (CD56+CD8α−) cells following treatmentwith IL-15/Rα variants.

FIG. 60A-60D depicts the percentage of Ki67 expression on (A) CD8⁺ Tcells, (B) CD4⁺ T cells, (C) γδ T cells and (D) NK (CD16⁺) cellsfollowing treatment with additional IL-15/Rα variants.

FIG. 61A-61D depicts the percentage of Ki67 expression on (A) CD8⁺ Tcells, (B) CD4⁺ T cells, (C) γδ T cells and (D) NK (CD16⁺) cellsfollowing treatment with additional IL-15/Rα variants.

FIG. 62 depicts IV-TV Dose PK of various IL-15/Rα Fc fusion proteins orcontrols in C57BL/6 mice at 0.1 mg/kg single dose

FIG. 63 depicts the correlation of half-life vs NK cell potencyfollowing treatment with IL-15/Rα-Fc fusion proteins engineered forlower potency.

FIG. 64A-64K depicts several formats for the IL-15/Rα×anti-PD-1bifunctional proteins of the present invention. The “scIL-15/Rα×scFv”format (FIG. 64A) comprises IL-15Rα(sushi) fused to IL-15 by a variablelength linker (termed “scIL-15/Rα”) which is then fused to theN-terminus of a heterodimeric Fc-region, with an scFv fused to the otherside of the heterodimeric Fc. The “scFv×ncIL-15/Rα” format (FIG. 64B)comprises an scFv fused to the N-terminus of a heterodimeric Fc-region,with IL-15Rα(sushi) fused to the other side of the heterodimeric Fc,while IL-15 is transfected separately so that a non-covalent IL-15/Rαcomplex is formed. The “scFv×dsIL-15/Rα” format (FIG. 64C) is the sameas the “scFv×ncIL-15/Rα” format, but wherein IL-15Rα(sushi) and IL-15are covalently linked as a result of engineered cysteines. The“scIL-15/Rα×Fab” format (FIG. 64D) comprises IL-15Rα(sushi) fused toIL-15 by a variable length linker (termed “scIL-15/Rα”) which is thenfused to the N-terminus of a heterodimeric Fc-region, with a variableheavy chain (VH) fused to the other side of the heterodimeric Fc, whilea corresponding light chain is transfected separately so as to form aFab with the VH. The “ncIL-15/Rα×Fab” format (FIG. 64E) comprises a VHfused to the N-terminus of a heterodimeric Fc-region, withIL-15Rα(sushi) fused to the other side of the heterodimeric Fc, while acorresponding light chain is transfected separately so as to form a Fabwith the VH, and while IL-15 is transfected separately so that anon-covalent IL-15/Rα complex is formed. The “dsIL-15/Rα×Fab” format(FIG. 64F) is the same as the “ncIL-15/Rα×Fab” format, but whereinIL-15Rα(sushi) and IL-15 are covalently linked as a result of engineeredcysteines. The “mAb-scIL-15/Rα” format (FIG. 64G) comprises VH fused tothe N-terminus of a first and a second heterodimeric Fc, with IL-15 isfused to IL-15Rα(sushi) which is then further fused to the C-terminus ofone of the heterodimeric Fc-region, while corresponding light chains aretransfected separately so as to form a Fabs with the VHs. The“mAb-ncIL-15/Rα” format (FIG. 64H) comprises VH fused to the N-terminusof a first and a second heterodimeric Fc, with IL-15Rα(sushi) fused tothe C-terminus of one of the heterodimeric Fc-region, whilecorresponding light chains are transfected separately so as to form aFabs with the VHs, and while and while IL-15 is transfected separatelyso that a non-covalent IL-15/Rα complex is formed. The “mAb-dsIL-15/Rα”format (FIG. 64I) is the same as the “mAb-ncIL-15/Rα” format, butwherein IL-15Rα(sushi) and IL-15 are covalently linked as a result ofengineered cysteines. The “central-IL-15/Rα” format (FIG. 64J) comprisesa VH recombinantly fused to the N-terminus of IL-15 which is thenfurther fused to one side of a heterodimeric Fc and a VH recombinantlyfused to the N-terminus of IL-15Rα(sushi) which is then further fused tothe other side of the heterodimeric Fc, while corresponding light chainsare transfected separately so as to form a Fabs with the VHs. The“central-scIL-15/Rα” format (FIG. 64K) comprises a VH fused to theN-terminus of IL-15Rα(sushi) which is fused to IL-15 which is thenfurther fused to one side of a heterodimeric Fc and a VH fused to theother side of the heterodimeric Fc, while corresponding light chains aretransfected separately so as to form a Fabs with the VHs.

FIG. 65 depicts sequences of XENP21480, an illustrativeIL-15/Rα×anti-PD-1 bifunctional protein of the “scIL-15/Rα×scFv” format.The CDRs are in bold. As noted herein and is true for every sequenceherein containing CDRs, the exact identification of the CDR locationsmay be slightly different depending on the numbering used as is shown inTable 1, and thus included herein are not only the CDRs that areunderlined but also CDRs included within the VH and VL domains usingother numbering systems. IL-15 and IL-15Rα(sushi) are underlined,linkers are double underlined (although as will be appreciated by thosein the art, the linkers can be replaced by other linkers, some of whichare depicted in FIGS. 8 and 9A-9C), and slashes (/) indicate theborder(s) between IL-15, IL-15Rα, linkers, variable regions, andconstant/Fc regions.

FIG. 66 depicts sequences of an illustrative IL-15/Rα×anti-PD-1bifunctional protein of the “scFv×ncIL-15/Rα” format. The CDRs are inbold. As noted herein and is true for every sequence herein containingCDRs, the exact identification of the CDR locations may be slightlydifferent depending on the numbering used as is shown in Table 1, andthus included herein are not only the CDRs that are underlined but alsoCDRs included within the VH and VL domains using other numberingsystems. IL-15 and IL-15Rα(sushi) are underlined, linkers are doubleunderlined (although as will be appreciated by those in the art, thelinkers can be replaced by other linkers, some of which are depicted inFIGS. 8 and 9A-9C), and slashes (I) indicate the border(s) betweenIL-15, IL-15Rα, linkers, variable regions, and constant/Fc regions.

FIG. 67 depicts sequences of an illustrative IL-15/Rα×anti-PD-1bifunctional protein of the “scFv×dsIL-15/Rα” format. The CDRs are inbold. As noted herein and is true for every sequence herein containingCDRs, the exact identification of the CDR locations may be slightlydifferent depending on the numbering used as is shown in Table 1, andthus included herein are not only the CDRs that are underlined but alsoCDRs included within the VH and VL domains using other numberingsystems. IL-15 and IL-15Rα(sushi) are underlined, linkers are doubleunderlined (although as will be appreciated by those in the art, thelinkers can be replaced by other linkers, some of which are depicted inFIGS. 8 and 9A-9C), and slashes (I) indicate the border(s) betweenIL-15, IL-15Rα, linkers, variable regions, and constant/Fc regions.

FIG. 68A-68C depicts sequences of illustrative IL-15/Rα×anti-PD-1bifunctional proteins of the “scIL-15/Rα×Fab” format. The CDRs are inbold. As noted herein and is true for every sequence herein containingCDRs, the exact identification of the CDR locations may be slightlydifferent depending on the numbering used as is shown in Table 1, andthus included herein are not only the CDRs that are underlined but alsoCDRs included within the VH and VL domains using other numberingsystems. IL-15 and IL-15Rα(sushi) are underlined, linkers are doubleunderlined (although as will be appreciated by those in the art, thelinkers can be replaced by other linkers, some of which are depictedFIGS. 8 and 9A-9C), and slashes (I) indicate the border(s) betweenIL-15, IL-15Rα, linkers, variable regions, and constant/Fc regions.

FIG. 69 depicts sequences of XENP22112, an illustrativeIL-15/Rα×anti-PD-1 bifunctional protein of the “Fab×ncIL-15/Rα” format.The CDRs are in bold. As noted herein and is true for every sequenceherein containing CDRs, the exact identification of the CDR locationsmay be slightly different depending on the numbering used as is shown inTable 1, and thus included herein are not only the CDRs that areunderlined but also CDRs included within the VH and VL domains usingother numbering systems. IL-15 and IL-15Rα(sushi) are underlined,linkers are double underlined (although as will be appreciated by thosein the art, the linkers can be replaced by other linkers, some of whichare depicted in FIGS. 8 and 9A-9C), and slashes (/) indicate theborder(s) between IL-15, IL-15Rα, linkers, variable regions, andconstant/Fc regions.

FIG. 70 depicts sequences of XENP22641, an illustrativeIL-15/Rα×anti-PD-1 bifunctional protein of the “Fab×dsIL-15/Rα” format.The CDRs are in bold. As noted herein and is true for every sequenceherein containing CDRs, the exact identification of the CDR locationsmay be slightly different depending on the numbering used as is shown inTable 1, and thus included herein are not only the CDRs that areunderlined but also CDRs included within the VH and VL domains usingother numbering systems. IL-15 and IL-15Rα(sushi) are underlined,linkers are double underlined (although as will be appreciated by thosein the art, the linkers can be replaced by other linkers, some of whichare depicted in FIGS. 8 and 9A-9C), and slashes (/) indicate theborder(s) between IL-15, IL-15Rα, linkers, variable regions, andconstant/Fc regions.

FIG. 71A-71B depicts sequences of an illustrative IL-15/Rα×anti-PD-1bifunctional protein of the “mAb×scIL-15/Rα” format. The CDRs are inbold. As noted herein and is true for every sequence herein containingCDRs, the exact identification of the CDR locations may be slightlydifferent depending on the numbering used as is shown in Table 1, andthus included herein are not only the CDRs that are underlined but alsoCDRs included within the VH and VL domains using other numberingsystems. IL-15 and IL-15Rα(sushi) are underlined, linkers are doubleunderlined (although as will be appreciated by those in the art, thelinkers can be replaced by other linkers, some of which are depicted inFigures FIGS. 8 and 9A-9C), and slashes (/) indicate the border(s)between IL-15, IL-15Rα, linkers, variable regions, and constant/Fcregions.

FIG. 72 depicts sequences of XENP22642 and XENP22643, illustrativeIL-15/Rα×anti-PD-1 bifunctional proteins of the “mAb×ncIL-15/Rα” format.The CDRs are in bold.

As noted herein and is true for every sequence herein containing CDRs,the exact identification of the CDR locations may be slightly differentdepending on the numbering used as is shown in Table 1, and thusincluded herein are not only the CDRs that are underlined but also CDRsincluded within the VH and VL domains using other numbering systems.IL-15 and IL-15Rα(sushi) are underlined, linkers are double underlined(although as will be appreciated by those in the art, the linkers can bereplaced by other linkers, some of which are depicted in FIGS. 8 and9A-9C), and slashes (/) indicate the border(s) between IL-15, IL-15Rα,linkers, variable regions, and constant/Fc regions.

FIG. 73 depicts sequences of XENP22644 and XENP22645, illustrativeIL-15/Rα×anti-PD-1 bifunctional proteins of the “mAb×dsIL-15/Rα” format.The CDRs are in bold. As noted herein and is true for every sequenceherein containing CDRs, the exact identification of the CDR locationsmay be slightly different depending on the numbering used as is shown inTable 1, and thus included herein are not only the CDRs that areunderlined but also CDRs included within the VH and VL domains usingother numbering systems. IL-15 and IL-15Rα(sushi) are underlined,linkers are double underlined (although as will be appreciated by thosein the art, the linkers can be replaced by other linkers, some of whichare depicted in FIGS. 8 and 9A-9C), and slashes (/) indicate theborder(s) between IL-15, IL-15Rα, linkers, variable regions, andconstant/Fc regions.

FIG. 74 depicts sequences of illustrative IL-15/Rα×anti-PD-1bifunctional proteins of the “central-IL-15/Rα” format. The CDRs are inbold. As noted herein and is true for every sequence herein containingCDRs, the exact identification of the CDR locations may be slightlydifferent depending on the numbering used as is shown in Table 1, andthus included herein are not only the CDRs that are underlined but alsoCDRs included within the VH and VL domains using other numberingsystems. IL-15 and IL-15Rα(sushi) are underlined, linkers are doubleunderlined (although as will be appreciated by those in the art, thelinkers can be replaced by other linkers, some of which are depicted inFIGS. 8 and 9A-9C), and slashes (/) indicate the border(s) betweenIL-15, IL-15Rα, linkers, variable regions, and constant/Fc regions.

FIG. 75 depicts sequences of illustrative IL-15/Rα×anti-PD-1bifunctional proteins of the “central-scIL-15/Rα” format. The CDRs arein bold. As noted herein and is true for every sequence hereincontaining CDRs, the exact identification of the CDR locations may beslightly different depending on the numbering used as is shown in Table1, and thus included herein are not only the CDRs that are underlinedbut also CDRs included within the V_(H) and V_(L) domains using othernumbering systems. IL-15 and IL-15Rα(sushi) are underlined, linkers aredouble underlined (although as will be appreciated by those in the art,the linkers can be replaced by other linkers, some of which are depictedin FIGS. 8 and 9A-9C), and slashes (/) indicate the border(s) betweenIL-15, IL-15Rα, linkers, variable regions, and constant/Fc regions.

FIG. 76A-76F depicts A) the format for illustrative IL-15/Rα×anti-PD-1bifunctional protein XENP21480, the purity and homogeneity of XENP21480as determined by B) SEC and C) CEF, the affinity of XENP21480 for D)IL-2RB and E) PD-1 as determined by Octet, and F) the stability ofXENP21480 as determined by DSF.

FIG. 77A-77B depicts the sensorgrams from Octet experiment forconfirming the binding of two batches of XENP25850 to A) IL-2RB:commongamma chain complex and B) PD-1.

FIG. 78A-78C depicts the induction of A) NK (CD56⁺/CD16⁺) cells, B) CD4⁺T cells, and C) CD8⁺ T cells proliferation by illustrativeIL-15/Rα×anti-PD-1 bifunctional protein and controls.

FIG. 79 depicts enhancement of IL-2 secretion by an illustrativeIL-15/Rα×anti-PD-1 bifunctional protein and controls over PBS in anSEB-stimulated PBMC assay.

FIG. 80 depicts IFNγ level on Days 4, 7, and 11 in serum of huPBMCengrafted mice following treatment with an illustrativeIL-15/Rα×anti-PD-1 bifunctional protein XENP25850 and controls.

FIG. 81A-81C depicts CD8⁺ T cell count on Days A) 4, B) 7, and C) 11 inwhole blood of huPBMC engrafted mice following treatment with anillustrative IL-15/Rα×anti-PD-1 bifunctional protein XENP25850 andcontrols.

FIG. 82A-82C depicts CD4⁺ T cell count on Days A) 4, B) 7, and C) 11 inwhole blood of huPBMC engrafted mice following treatment with anillustrative IL-15/Rα× anti-PD-1 bifunctional protein XENP25850 andcontrols.

FIG. 83A-83C depicts CD45+ cell count on Days A) 4, B) 7, and C) 11 inwhole blood of huPBMC engrafted mice following treatment with anillustrative IL-15/Rα×anti-PD-1 bifunctional protein XENP25850 andcontrols.

FIG. 84A-84C depicts the body weight as a percentage of initial bodyweight of huPBMC engrafted mice on Days A) 4, B) 7, and C) 11 followingtreatment with an illustrative IL-15/Rα×anti-PD-1 bifunctional proteinXENP25850 and controls. Each point represents a single NSG mouse. Micewhose body weights dropped below 70% initial body weight wereeuthanized. Dead mice are represented as 70%.

FIG. 85A-85N depicts sequences of the invention. The CDRs are in bold,IL-15 and IL15-Rα(sushi) are underlined, linkers are double underlined,and slashes (/) are between IL-15, IL15-Rα(sushi), linkers, and Fcdomains.

FIG. 86A-86F depicts additional anti-PD-1 Fv sequences from othersources that can find use in the present invention, either as a scFvconstruct (either in the vh-scFv linker-vl or vl-scFv linker-vhorientation) or as a Fab, and linked to the Fc domains as outlinedherein, using optional domain linkers. As for most of the sequencesdepicted herein, the CDRs are underlined.

FIG. 87A-87B depicts sequences of particular use in the presentinvention, with FIG. 87A depicting a scFv IL-15/Rα(sushi) construct andFIG. 87 b depicting the IL-15 and IL-15/Rα(sushi) with engineeredcysteines.

FIG. 88 depicts a list of engineered heterodimer-skewing (e.g. “stericheterodimerization”) Fc variants with heterodimer yields (determined byHPLC-CIEX) and thermal stabilities (determined by DSC). Not determinedthermal stability is denoted by “n.d.”.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

In order that the application may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “ablation” herein is meant a decrease or removal of activity. Thusfor example, “ablating FcγR binding” means the Fc region amino acidvariant has less than 50% starting binding as compared to an Fc regionnot containing the specific variant, with less than 70-80-90-95-98% lossof activity being preferred, and in general, with the activity beingbelow the level of detectable binding in a Biacore assay. Of particularuse in the ablation of FcγR binding are those shown in FIG. 3 . However,unless otherwise noted, the Fc monomers of the invention retain bindingto the FcRn receptor.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. ADCC is correlated withbinding to FcγRIIIa; increased binding to FcγRIIIa leads to an increasein ADCC activity. As is discussed herein, many embodiments of theinvention ablate ADCC activity entirely.

By “ADCP” or antibody dependent cell-mediated phagocytosis as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause phagocytosis of the target cell.

By “antigen binding domain” or “ABD” herein is meant a set of sixComplementary Determining Regions (CDRs) that, when present as part of apolypeptide sequence, specifically binds a target antigen as discussedherein. Thus, a “checkpoint antigen binding domain” binds a targetcheckpoint antigen as outlined herein. As is known in the art, theseCDRs are generally present as a first set of variable heavy CDRs (vhCDRsor VHCDRs) and a second set of variable light CDRs (vlCDRs or VLCDRs),each comprising three CDRs: vhCDR1, vhCDR2, vhCDR3 for the heavy chainand vlCDR1, vlCDR2 and vlCDR3 for the light. The CDRs are present in thevariable heavy and variable light domains, respectively, and togetherform an Fv region. Thus, in some cases, the six CDRs of the antigenbinding domain are contributed by a variable heavy and variable lightchain. In a “Fab” format, the set of 6 CDRs are contributed by twodifferent polypeptide sequences, the variable heavy domain (vh or VH;containing the vhCDR1, vhCDR2 and vhCDR3) and the variable light domain(vl or V_(L); containing the vlCDR1, vlCDR2 and vlCDR3), with theC-terminus of the vh domain being attached to the N-terminus of the CH1domain of the heavy chain and the C-terminus of the vl domain beingattached to the N-terminus of the constant light domain (and thusforming the light chain). In a scFv format, the vh and vl domains arecovalently attached, generally through the use of a linker as outlinedherein, into a single polypeptide sequence, which can be either(starting from the N-terminus) vh-linker-vl or vl-linker-vh, with theformer being generally preferred (including optional domain linkers oneach side, depending on the format used (e.g., from FIG. 1 of U.S.62/353,511).

By “modification” herein is meant an amino acid substitution, insertion,and/or deletion in a polypeptide sequence or an alteration to a moietychemically linked to a protein. For example, a modification may be analtered carbohydrate or PEG structure attached to a protein. By “aminoacid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. For clarity,unless otherwise noted, the amino acid modification is always to anamino acid coded for by DNA, e.g., the 20 amino acids that have codonsin DNA and RNA.

By “amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with a different amino acid. In particular, in someembodiments, the substitution is to an amino acid that is not naturallyoccurring at the particular position, either not naturally occurringwithin the organism or in any organism. For example, the substitutionE272Y refers to a variant polypeptide, in this case an Fc variant, inwhich the glutamic acid at position 272 is replaced with tyrosine. Forclarity, a protein which has been engineered to change the nucleic acidcoding sequence but not change the starting amino acid (for exampleexchanging CGG (encoding arginine) to CGA (still encoding arginine) toincrease host organism expression levels) is not an “amino acidsubstitution”; that is, despite the creation of a new gene encoding thesame protein, if the protein has the same amino acid at the particularposition that it started with, it is not an amino acid substitution.

By “amino acid insertion” or “insertion” as used herein is meant theaddition of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, −233E or 233E designates an insertionof glutamic acid after position 233 and before position 234.Additionally, −233ADE or A233ADE designates an insertion of AlaAspGluafter position 233 and before position 234.

By “amino acid deletion” or “deletion” as used herein is meant theremoval of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, E233− or E233#, E233( ) or E233deldesignates a deletion of glutamic acid at position 233. Additionally,EDA233− or EDA233# designates a deletion of the sequence GluAspAla thatbegins at position 233.

By “variant protein” or “protein variant”, or “variant” as used hereinis meant a protein that differs from that of a parent protein by virtueof at least one amino acid modification. Protein variant may refer tothe protein itself, a composition comprising the protein, or the aminosequence that encodes it. Preferably, the protein variant has at leastone amino acid modification compared to the parent protein, e.g. fromabout one to about seventy amino acid modifications, and preferably fromabout one to about five amino acid modifications compared to the parent.As described below, in some embodiments the parent polypeptide, forexample an Fc parent polypeptide, is a human wild type sequence, such asthe Fc region from IgG1, IgG2, IgG3 or IgG4, although human sequenceswith variants can also serve as “parent polypeptides”, for example theIgG1/2 hybrid can be included. The protein variant sequence herein willpreferably possess at least about 80% identity with a parent proteinsequence, and most preferably at least about 90% identity, morepreferably at least about 95-98-99% identity. Variant protein can referto the variant protein itself, compositions comprising the proteinvariant, or the DNA sequence that encodes it.

Accordingly, by “antibody variant” or “variant antibody” as used hereinis meant an antibody that differs from a parent antibody by virtue of atleast one amino acid modification, “IgG variant” or “variant IgG” asused herein is meant an antibody that differs from a parent IgG (again,in many cases, from a human IgG sequence) by virtue of at least oneamino acid modification, and “immunoglobulin variant” or “variantimmunoglobulin” as used herein is meant an immunoglobulin sequence thatdiffers from that of a parent immunoglobulin sequence by virtue of atleast one amino acid modification. “Fc variant” or “variant Fc” as usedherein is meant a protein comprising an amino acid modification in an Fcdomain. The Fc variants of the present invention are defined accordingto the amino acid modifications that compose them. Thus, for example,N434S or 434S is an Fc variant with the substitution serine at position434 relative to the parent Fc polypeptide, wherein the numbering isaccording to the EU index. Likewise, M428L/N434S defines an Fc variantwith the substitutions M428L and N434S relative to the parent Fcpolypeptide. The identity of the WT amino acid may be unspecified, inwhich case the aforementioned variant is referred to as 428L/434S. It isnoted that the order in which substitutions are provided is arbitrary,that is to say that, for example, 428L/434S is the same Fc variant asM428L/N434S, and so on. For all positions discussed in the presentinvention that relate to antibodies, unless otherwise noted, amino acidposition numbering is according to the EU index. The EU index or EUindex as in Kabat or EU numbering scheme refers to the numbering of theEU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85,hereby entirely incorporated by reference.) The modification can be anaddition, deletion, or substitution. Substitutions can include naturallyoccurring amino acids and, in some cases, synthetic amino acids.Examples include U.S. Pat. No. 6,586,207; WO 98/48032; WO 03/073238;US2004-0214988A1; WO 05/35727A2; WO 05/74524A2; J. W. Chin et al.,(2002), Journal of the American Chemical Society 124:9026-9027; J. W.Chin, & P. G. Schultz, (2002), ChemBioChem 11:1135-1137; J. W. Chin, etal., (2002), PICAS United States of America 99:11020-11024; and, L.Wang, & P. G. Schultz, (2002), Chem. 1-10, all entirely incorporated byreference.

As used herein, “protein” herein is meant at least two covalentlyattached amino acids, which includes proteins, polypeptides,oligopeptides and peptides. The peptidyl group may comprise naturallyoccurring amino acids and peptide bonds, or synthetic peptidomimeticstructures, i.e. “analogs”, such as peptoids (see Simon et al., PNAS USA89(20):9367 (1992), entirely incorporated by reference). The amino acidsmay either be naturally occurring or synthetic (e.g. not an amino acidthat is coded for by DNA); as will be appreciated by those in the art.For example, homo-phenylalanine, citrulline, ornithine and noreleucineare considered synthetic amino acids for the purposes of the invention,and both D- and L-(R or S) configured amino acids may be utilized. Thevariants of the present invention may comprise modifications thatinclude the use of synthetic amino acids incorporated using, forexample, the technologies developed by Schultz and colleagues, includingbut not limited to methods described by Cropp & Shultz, 2004, TrendsGenet. 20(12):625-30, Anderson et al., 2004, Proc Natl Acad Sci USA 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin et al., 2003,Science 301(5635):964-7, all entirely incorporated by reference. Inaddition, polypeptides may include synthetic derivatization of one ormore side chains or termini, glycosylation, PEGylation, circularpermutation, cyclization, linkers to other molecules, fusion to proteinsor protein domains, and addition of peptide tags or labels.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297 or N297) is a residue at position 297 in the humanantibody IgG.

By “Fab” or “Fab region” as used herein is meant the polypeptide thatcomprises the VH, CH1, VL, and CL immunoglobulin domains. Fab may referto this region in isolation, or this region in the context of a fulllength antibody, antibody fragment or Fab fusion protein.

By “Fv” or “Fv fragment” or “Fv region” as used herein is meant apolypeptide that comprises the VL and VH domains of a single antibody.As will be appreciated by those in the art, these generally are made upof two chains, or can be combined (generally with a linker as discussedherein) to form an scFv.

By “single chain Fv” or “scFv” herein is meant a variable heavy domaincovalently attached to a variable light domain, generally using a scFvlinker as discussed herein, to form a scFv or scFv domain. A scFv domaincan be in either orientation from N to C-terminus (vh-linker-vl orvl-linker-vh).

By “IgG subclass modification” or “isotype modification” as used hereinis meant an amino acid modification that converts one amino acid of oneIgG isotype to the corresponding amino acid in a different, aligned IgGisotype. For example, because IgG1 comprises a tyrosine and IgG2 aphenylalanine at EU position 296, a F296Y substitution in IgG2 isconsidered an IgG subclass modification.

By “non-naturally occurring modification” as used herein is meant anamino acid modification that is not isotypic. For example, because noneof the IgGs comprise a serine at position 434, the substitution 434S inIgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered anon-naturally occurring modification.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids that are coded for by DNA andRNA.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toADCC, ADCP, and CDC.

By “IgG Fc ligand” as used herein is meant a molecule, preferably apolypeptide, from any organism that binds to the Fc region of an IgGantibody to form an Fc/Fc ligand complex. Fc ligands include but are notlimited to FcγRIs, FcγRIIs, FcγRIIIs, FcRn, C1q, C3, mannan bindinglectin, mannose receptor, staphylococcal protein A, streptococcalprotein G, and viral FcγR. Fc ligands also include Fc receptor homologs(FcRH), which are a family of Fc receptors that are homologous to theFcγRs (Davis et al., 2002, Immunological Reviews 190:123-136, entirelyincorporated by reference). Fc ligands may include undiscoveredmolecules that bind Fc. Particular IgG Fc ligands are FcRn and Fc gammareceptors. By “Fc ligand” as used herein is meant a molecule, preferablya polypeptide, from any organism that binds to the Fc region of anantibody to form an Fc/Fc ligand complex.

By “Fc gamma receptor”, “FcγR” or “FcgammaR” as used herein is meant anymember of the family of proteins that bind the IgG antibody Fc regionand is encoded by an FcγR gene. In humans this family includes but isnot limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, andFcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypesH131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), andFcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirelyincorporated by reference), as well as any undiscovered human FcγRs orFcγR isoforms or allotypes. An FcγR may be from any organism, includingbut not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRsinclude but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII(CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRsor FcγR isoforms or allotypes.

By “FcRn” or “neonatal Fc Receptor” as used herein is meant a proteinthat binds the IgG antibody Fc region and is encoded at least in part byan FcRn gene. The FcRn may be from any organism, including but notlimited to humans, mice, rats, rabbits, and monkeys. As is known in theart, the functional FcRn protein comprises two polypeptides, oftenreferred to as the heavy chain and light chain. The light chain isbeta-2-microglobulin and the heavy chain is encoded by the FcRn gene.Unless otherwise noted herein, FcRn or an FcRn protein refers to thecomplex of FcRn heavy chain with beta-2-microglobulin. A variety of FcRnvariants can be used to increase binding to the FcRn receptor, and insome cases, to increase serum half-life. In general, unless otherwisenoted, the Fc monomers of the invention retain binding to the FcRnreceptor (and, as noted below, can include amino acid variants toincrease binding to the FcRn receptor).

By “parent polypeptide” as used herein is meant a starting polypeptidethat is subsequently modified to generate a variant. The parentpolypeptide may be a naturally occurring polypeptide, or a variant orengineered version of a naturally occurring polypeptide. Parentpolypeptide may refer to the polypeptide itself, compositions thatcomprise the parent polypeptide, or the amino acid sequence that encodesit. Accordingly, by “parent immunoglobulin” as used herein is meant anunmodified immunoglobulin polypeptide that is modified to generate avariant, and by “parent antibody” as used herein is meant an unmodifiedantibody that is modified to generate a variant antibody. It should benoted that “parent antibody” includes known commercial, recombinantlyproduced antibodies as outlined below.

By “Fc” or “Fc region” or “Fc domain” as used herein is meant thepolypeptide comprising the constant region of an antibody excluding thefirst constant region immunoglobulin domain (e.g., CH1) and in somecases, part of the hinge. Thus Fc refers to the last two constant regionimmunoglobulin domains (e.g., CH2 and CH3) of IgA, IgD, and IgG, thelast three constant region immunoglobulin domains of IgE and IgM, andthe flexible hinge N-terminal to these domains. For IgA and IgM, Fc mayinclude the J chain. For IgG, the Fc domain comprises immunoglobulindomains Cγ2 and Cγ3 (Cγ2 and Cγ3) and the lower hinge region between Cγ1(Cγ1) and Cγ2 (Cγ2). Although the boundaries of the Fc region may vary,the human IgG heavy chain Fc region is usually defined to includeresidues C226 or P230 to its carboxyl-terminus, wherein the numbering isaccording to the EU index as in Kabat. In some embodiments, as is morefully described below, amino acid modifications are made to the Fcregion, for example to alter binding to one or more FcγR receptors or tothe FcRn receptor.

By “heavy constant region” herein is meant the CH1-hinge-CH2-CH3 portionof an antibody.

By “Fc fusion protein” or “immunoadhesin” herein is meant a proteincomprising an Fc region, generally linked (optionally through a linkermoiety, as described herein) to a different protein, such as to IL-15and/or IL-15R, as described herein. In some instances, two Fc fusionproteins can form a homodimeric Fc fusion protein or a heterodimeric Fcfusion protein with the latter being preferred. In some cases, onemonomer of the heterodimeric Fc fusion protein comprises an Fc domainalone (e.g., an empty Fc domain) and the other monomer is a Fc fusion,comprising a variant Fc domain and a protein domain, such as a receptor,ligand or other binding partner.

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index for antibody numbering.

By “strandedness” in the context of the monomers of the heterodimericantibodies of the invention herein is meant that, similar to the twostrands of DNA that “match”, heterodimerization variants areincorporated into each monomer so as to preserve the ability to “match”to form heterodimers. For example, if some pI variants are engineeredinto monomer A (e.g., making the pI higher) then steric variants thatare “charge pairs” that can be utilized as well do not interfere withthe pI variants, e.g., the charge variants that make a pI higher are puton the same “strand” or “monomer” to preserve both functionalities.Similarly, for “skew” variants that come in pairs of a set as more fullyoutlined below, the skilled artisan will consider pI in deciding intowhich strand or monomer that incorporates one set of the pair will go,such that pI separation is maximized using the pI of the skews as well.

By “target antigen” as used herein is meant the molecule that is boundspecifically by the variable region of a given antibody. A targetantigen may be a protein, carbohydrate, lipid, or other chemicalcompound. A wide number of suitable target antigens are described below.

By “target cell” as used herein is meant a cell that expresses a targetantigen.

By “variable region” as used herein is meant the region of animmunoglobulin that comprises one or more Ig domains substantiallyencoded by any of the Vκ, Vλ, and/or VH genes that make up the kappa,lambda, and heavy chain immunoglobulin genetic loci respectively.

By “wild type or WT” herein is meant an amino acid sequence or anucleotide sequence that is found in nature, including allelicvariations. A WT protein has an amino acid sequence or a nucleotidesequence that has not been intentionally modified.

The biospecific heterodimeric proteins of the present invention aregenerally isolated or recombinant. “Isolated,” when used to describe thevarious polypeptides disclosed herein, means a polypeptide that has beenidentified and separated and/or recovered from a cell or cell culturefrom which it was expressed. Ordinarily, an isolated polypeptide will beprepared by at least one purification step. An “isolated protein,”refers to a protein which is substantially free of other proteins havingdifferent binding specificities. “Recombinant” means the proteins aregenerated using recombinant nucleic acid techniques in exogeneous hostcells.

“Percent (%) amino acid sequence identity” with respect to a proteinsequence is defined as the percentage of amino acid residues in acandidate sequence that are identical with the amino acid residues inthe specific (parental) sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.One particular program is the ALIGN-2 program outlined at paragraphs[0279] to [0280] of US Pub. No. 20160244525, hereby incorporated byreference.

The degree of identity between an amino acid sequence of the presentinvention (“invention sequence”) and the parental amino acid sequence iscalculated as the number of exact matches in an alignment of the twosequences, divided by the length of the “invention sequence,” or thelength of the parental sequence, whichever is the shortest. The resultis expressed in percent identity.

In some embodiments, two or more amino acid sequences are at least 50%,60%, 70%, 80%, or 90% identical. In some embodiments, two or more aminoacid sequences are at least 95%, 97%, 98%, 99%, or even 100% identical.

“Specific binding” or “specifically binds to” or is “specific for” aparticular antigen or an epitope means binding that is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity. For example, specificbinding can be determined by competition with a control molecule that issimilar to the target.

Specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KD for an antigen orepitope of at least about 10⁻⁴ M, at least about 10⁻⁵ M, at least about10⁻⁶ M, at least about 10⁻⁷ M, at least about 10⁻⁸ M, at least about10⁻⁹ M, alternatively at least about 10⁻¹⁰ M, at least about 10⁻¹¹ M, atleast about 10⁻¹² M, or greater, where KD refers to a dissociation rateof a particular antibody-antigen interaction. Typically, an antibodythat specifically binds an antigen will have a KD that is 20-, 50-,100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a controlmolecule relative to the antigen or epitope.

Also, specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KA or Ka for an antigenor epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- ormore times greater for the epitope relative to a control, where KA or Karefers to an association rate of a particular antibody-antigeninteraction. Binding affinity is generally measured using a Biacoreassay.

II. Introduction

The invention provides heterodimeric fusion proteins that can bind tothe checkpoint inhibitor PD-1 antigen and can complex with the commongamma chain (γc; CD132) and/or the Il-2 receptor β-chain (IL-2Rβ;CD122). In general, the heterodimeric fusion proteins of the inventionhave three functional components: an IL-15/IL-15Rα(sushi) component,generally referred to herein as an “IL-15 complex”, an anti-PD-1component, and an Fc component, each of which can take different formsand each of which can be combined with the other components in anyconfiguration.

A. IL-15/IL-15Rα(Sushi) Domains

As shown in the figures, the IL-15 complex can take several forms. Asstated above, the IL-15 protein on its own is less stable than whencomplexed with the IL-15Rα protein. As is known in the art, the IL-15Rαprotein contains a “sushi domain”, which is the shortest region of thereceptor that retains IL-15 binding activity. Thus, while heterodimericfusion proteins comprising the entire IL-15Rα protein can be made,preferred embodiments herein include complexes that just use the sushidomain, the sequence of which is shown in the figures.

Accordingly, the IL-15 complex generally comprises the IL-15 protein andthe sushi domain of IL IL-15Rα (unless otherwise noted that the fulllength sequence is used, “IL-15Rα”, “IL-15Rα(sushi)” and “sushi” areused interchangeably throughout). This complex can be used in threedifferent formats. As shown in FIG. 64B, the IL-15 protein and theIL-15Rα(sushi) are not covalently attached, but rather areself-assembled through regular ligand-ligand interactions. As is morefully described herein, it can be either the IL-15 domain or the sushidomain that is covalently linked to the Fc domain (generally using anoptional domain linker). Alternatively, they can be covalently attachedusing a domain linker as generally shown in FIG. 64A. FIG. 64A depictsthe sushi domain as the N-terminal domain, although this can bereversed. Finally, each of the IL-15 and sushi domains can be engineeredto contain a cysteine amino acid, that forms a disulfide bond to formthe complex as is generally shown in FIG. 64C, again, with either theIL-15 domain or the sushi domain being covalently attached (using anoptional domain linker) to the Fc domain.

B. Anti-PD-1 Components

The anti-PD-1 component (the anti-PD-1 antigen binding domain or ABD) ofthe invention is generally a set of 6 CDRs and/or a variable heavydomain and a variable light domain that form an Fv domain that can bindhuman PD-1. As shown herein, the anti-PD-1 ABD can be in the form of ascFv, wherein the vh and vl domains are joined using an scFv linker,which can be optionally a charged scFv linker. As will be appreciated bythose in the art, the scFv can be assembled from N- to C-terminus asN-vh-scFv linker-vl-C or as N-vl-scFv linker-vh-C, with the C terminusof the scFv domain generally being linked to the hinge-CH2-CH3 Fcdomain. Suitable Fvs (including CDR sets and variable heavy/variablelight domains) can be used in scFv formats or Fab formats are shown inthe Figures as well as disclosed in U.S. 62/353,511, the contents arehereby incorporated in its entirety for all purposes, and in particularfor the FIGS. 11 and 12 sequences.

As will further be appreciated by those in the art, all or part of thehinge (which can also be a wild type hinge from IgG1, IgG2 or IgG4 or avariant thereof, such as the IgG4 S241P or S228P hinge variant with thesubstitution proline at position 228 relative to the parent IgG4 hingepolypeptide (wherein the numbering S228P is according to the EU indexand the S241P is the Kabat numbering)) can be used as the domain linkerbetween the scFv and the CH2-CH3 domain, or a different domain linkersuch as depicted in the Figures can be used.

C. Fc Domains

The Fc domain component of the invention is as described herein, whichgenerally contains skew variants and/or optional pI variants and/orablation variants are outlined herein.

III. Bispecific IL-15/IL-15Rα Fc Fusion×PD-1 ABD Heterodimeric Proteins

Provided herein are heterodimeric fusion proteins that can bind to thecheckpoint inhibitor PD-1 antigen and can complex with the common gammachain (γc; CD132) and/or the 11-2 receptor β-chain (IL-2Rβ; CD122). Theheterodimeric fusion proteins can contain an IL-15/IL-15Rα-Fc fusionprotein and an antibody fusion protein. The IL-15/IL-15Rα-Fc fusionprotein can include as IL-15 protein covalently attached to an IL-15Rα,and an Fc domain. Optionally, the IL-15 protein and IL-15Rα protein arenoncovalently attached.

The Fc domains can be derived from IgG Fc domains, e.g., IgG1, IgG2,IgG3 or IgG4 Fc domains, with IgG1 Fc domains finding particular use inthe invention. The following describes Fc domains that are useful forIL-15/IL-15Rα Fc fusion monomers and checkpoint antibody fragments ofthe bispecific heterodimer proteins of the present invention.

The carboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function Kabat et al. collectednumerous primary sequences of the variable regions of heavy chains andlight chains. Based on the degree of conservation of the sequences, theyclassified individual primary sequences into the CDR and the frameworkand made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5thedition, NIH publication, No. 91-3242, E. A. Kabat et al., entirelyincorporated by reference). Throughout the present specification, theKabat numbering system is generally used when referring to a residue inthe variable domain (approximately, residues 1-107 of the light chainvariable region and residues 1-113 of the heavy chain variable region)and the EU numbering system for Fc regions (e.g., Kabat et al., supra(1991)).

In the IgG subclass of immunoglobulins, there are several immunoglobulindomains in the heavy chain. By “immunoglobulin (Ig) domain” herein ismeant a region of an immunoglobulin having a distinct tertiarystructure. Of interest in the present invention are the heavy chaindomains, including, the constant heavy (CH) domains and the hingedomains. In the context of IgG antibodies, the IgG isotypes each havethree CH regions. Accordingly, “CH” domains in the context of IgG are asfollows: “CH1” refers to positions 118-220 according to the EU index asin Kabat. “CH2” refers to positions 237-340 according to the EU index asin Kabat, and “CH3” refers to positions 341-447 according to the EUindex as in Kabat. As shown herein and described below, the pI variantscan be in one or more of the CH regions, as well as the hinge region,discussed below.

Another type of Ig domain of the heavy chain is the hinge region. By“hinge” or “hinge region” or “antibody hinge region” or “immunoglobulinhinge region” herein is meant the flexible polypeptide comprising theamino acids between the first and second constant domains of anantibody. Structurally, the IgG CH1 domain ends at EU position 220, andthe IgG CH2 domain begins at residue EU position 237. Thus for IgG theantibody hinge is herein defined to include positions 221 (D221 in IgG)to 236 (G236 in IgG1), wherein the numbering is according to the EUindex as in Kabat. In some embodiments, for example in the context of anFc region, the lower hinge is included, with the “lower hinge” generallyreferring to positions 226 or 230. As noted herein, pI variants can bemade in the hinge region as well.

Thus, the present invention provides different antibody domains, e.g.,different Fc domains. As described herein and known in the art, theheterodimeric proteins of the invention comprise different domains,which can be overlapping as well. These domains include, but are notlimited to, the Fc domain, the CH1 domain, the CH2 domain, the CH3domain, the hinge domain, and the heavy constant domain (CH1-hinge-Fcdomain or CH1-hinge-CH2-CH3).

Thus, the “Fc domain” includes the —CH2-CH3 domain, and optionally ahinge domain, and can be from human IgG1, IgG2, IgG3 or IgG4, with Fcdomains derived from IgG1. In some of the embodiments herein, when aprotein fragment, e.g., IL-15 or IL-15Rα is attached to an Fc domain, itis the C-terminus of the IL-15 or IL-15Rα construct that is attached toall or part of the hinge of the Fc domain; for example, it is generallyattached to the sequence EPKS (SEQ ID NO: 15) which is the beginning ofthe hinge. In other embodiments, when a protein fragment, e.g., IL-15 orIL-15Rα, is attached to an Fc domain, it is the C-terminus of the IL-15or IL-15Rα construct that is attached to the CH1 domain of the Fcdomain.

In some of the constructs and sequences outlined herein of an Fc domainprotein, the C-terminus of the IL-15 or IL-15Rα protein fragment isattached to the N-terminus of a domain linker, the C-terminus of whichis attached to the N-terminus of a constant Fc domain (N-IL-15 orIL-15Rα protein fragment-linker-Fc domain-C) although that can beswitched (N-Fc domain-linker-IL-15 or IL-15Rα protein fragment-C). Inother constructs and sequence outlined herein, C-terminus of a firstprotein fragment is attached to the N-terminus of a second proteinfragment, optionally via a domain linker, the C-terminus of the secondprotein fragment is attached to the N-terminus of a constant Fc domain,optionally via a domain linker. In yet other constructs and sequencesoutlined herein, a constant Fc domain that is not attached to a firstprotein fragment or a second protein fragment is provided. A heterodimerFc fusion protein can contain two or more of the exemplary monomeric Fcdomain proteins described herein.

In some embodiments, the linker is a “domain linker”, used to link anytwo domains as outlined herein together, some of which are depicted inFIG. 8 . While any suitable linker can be used, many embodiments utilizea glycine-serine polymer, including for example (GS)n (SEQ ID NO: 7),(GSGGS)n (SEQ ID NO: 8), (GGGGS)n (SEQ ID NO: 9), and (GGGS)n (SEQ IDNO: 10), where n is an integer of at least one (and generally from 1 to2 to 3 to 4 to 5) as well as any peptide sequence that allows forrecombinant attachment of the two domains with sufficient length andflexibility to allow each domain to retain its biological function. Insome cases, and with attention being paid to “strandedness”, as outlinedbelow, charged domain linkers.

In one embodiment, heterodimeric Fc fusion proteins contain at least twoconstant domains which can be engineered to produce heterodimers, suchas pI engineering. Other Fc domains that can be used include fragmentsthat contain one or more of the CH1, CH2, CH3, and hinge domains of theinvention that have been pI engineered. In particular, the formatsdepicted in FIG. 21 and FIG. 64 are heterodimeric Fc fusion proteins,meaning that the protein has two associated Fc sequences self-assembledinto a heterodimeric Fc domain and at least one protein fragment (e.g.,1, 2 or more protein fragments) as more fully described below. In somecases, a first protein fragment is linked to a first Fc sequence and asecond protein fragment is linked to a second Fc sequence. In othercases, a first protein fragment is linked to a first Fc sequence, andthe first protein fragment is non-covalently attached to a secondprotein fragment that is not linked to an Fc sequence. In some cases,the heterodimeric Fc fusion protein contains a first protein fragmentlinked to a second protein fragment which is linked a first Fc sequence,and a second Fc sequence that is not linked to either the first orsecond protein fragments.

Accordingly, in some embodiments the present invention providesheterodimeric Fc fusion proteins that rely on the use of two differentheavy chain variant Fc sequences, that will self-assemble to form aheterodimeric Fc domain fusion polypeptide.

The present invention is directed to novel constructs to provideheterodimeric Fc fusion proteins that allow binding to one or morebinding partners, ligands or receptors. The heterodimeric Fc fusionconstructs are based on the self-assembling nature of the two Fc domainsof the heavy chains of antibodies, e.g., two “monomers” that assembleinto a “dimer”. Heterodimeric Fc fusions are made by altering the aminoacid sequence of each monomer as more fully discussed below. Thus, thepresent invention is generally directed to the creation of heterodimericFc fusion proteins which can co-engage binding partner(s) or ligand(s)or receptor(s) in several ways, relying on amino acid variants in theconstant regions that are different on each chain to promoteheterodimeric formation and/or allow for ease of purification ofheterodimers over the homodimers.

There are a number of mechanisms that can be used to generate theheterodimers of the present invention. In addition, as will beappreciated by those in the art, these mechanisms can be combined toensure high heterodimerization. Thus, amino acid variants that lead tothe production of heterodimers are referred to as “heterodimerizationvariants”. As discussed below, heterodimerization variants can includesteric variants (e.g. the “knobs and holes” or “skew” variants describedbelow and the “charge pairs” variants described below) as well as “pIvariants”, which allows purification of homodimers away fromheterodimers. As is generally described in WO2014/145806, herebyincorporated by reference in its entirety and specifically as below forthe discussion of “heterodimerization variants”, useful mechanisms forheterodimerization include “knobs and holes” (“KIH”; sometimes herein as“skew” variants (see discussion in WO2014/145806), “electrostaticsteering” or “charge pairs” as described in WO2014/145806, pI variantsas described in WO2014/145806, and general additional Fc variants asoutlined in WO2014/145806 and below.

In the present invention, there are several basic mechanisms that canlead to ease of purifying heterodimeric antibodies; one relies on theuse of pI variants, such that each monomer has a different pI, thusallowing the isoelectric purification of A-A, A-B and B-B dimericproteins. Alternatively, some formats also allow separation on the basisof size. As is further outlined below, it is also possible to “skew” theformation of heterodimers over homodimers. Thus, a combination of stericheterodimerization variants and pI or charge pair variants findparticular use in the invention.

In general, embodiments of particular use in the present invention relyon sets of variants that include skew variants, that encourageheterodimerization formation over homodimerization formation, coupledwith pI variants, which increase the pI difference between the twomonomers.

Additionally, as more fully outlined below, depending on the format ofthe heterodimer Fc fusion protein, pI variants can be either containedwithin the constant and/or Fc domains of a monomer, or domain linkerscan be used. That is, the invention provides pI variants that are on oneor both of the monomers, and/or charged domain linkers as well. Inaddition, additional amino acid engineering for alternativefunctionalities may also confer pI changes, such as Fc, FcRn and KOvariants.

In the present invention that utilizes pI as a separation mechanism toallow the purification of heterodimeric proteins, amino acid variantscan be introduced into one or both of the monomer polypeptides; that is,the pI of one of the monomers (referred to herein for simplicity as“monomer A”) can be engineered away from monomer B, or both monomer Aand B change be changed, with the pI of monomer A increasing and the pIof monomer B decreasing. As discussed, the pI changes of either or bothmonomers can be done by removing or adding a charged residue (e.g., aneutral amino acid is replaced by a positively or negatively chargedamino acid residue, e.g., glycine to glutamic acid), changing a chargedresidue from positive or negative to the opposite charge (e.g. asparticacid to lysine) or changing a charged residue to a neutral residue(e.g., loss of a charge; lysine to serine). A number of these variantsare shown in the Figures.

Accordingly, this embodiment of the present invention provides forcreating a sufficient change in pI in at least one of the monomers suchthat heterodimers can be separated from homodimers. As will beappreciated by those in the art, and as discussed further below, thiscan be done by using a “wild type” heavy chain constant region and avariant region that has been engineered to either increase or decreaseits pI (wt A−+B or wt A−—B), or by increasing one region and decreasingthe other region (A+−B− or A−B+).

Thus, in general, a component of some embodiments of the presentinvention are amino acid variants in the constant regions that aredirected to altering the isoelectric point (pI) of at least one, if notboth, of the monomers of a dimeric protein by incorporating amino acidsubstitutions (“pI variants” or “pI substitutions”) into one or both ofthe monomers. As shown herein, the separation of the heterodimers fromthe two homodimers can be accomplished if the pIs of the two monomersdiffer by as little as 0.1 pH unit, with 0.2, 0.3, 0.4 and 0.5 orgreater all finding use in the present invention.

As will be appreciated by those in the art, the number of pI variants tobe included on each or both monomer(s) to get good separation willdepend in part on the starting pI of the components. As is known in theart, different Fcs will have different starting pIs which are exploitedin the present invention. In general, as outlined herein, the pIs areengineered to result in a total pI difference of each monomer of atleast about 0.1 logs, with 0.2 to 0.5 being preferred as outlinedherein.

As will be appreciated by those in the art, the number of pI variants tobe included on each or both monomer(s) to get good separation willdepend in part on the starting pI of the components. That is, todetermine which monomer to engineer or in which “direction” (e.g., morepositive or more negative), the sequences of the Fc domains, and in somecases, the protein domain(s) linked to the Fc domain are calculated anda decision is made from there. As is known in the art, different Fcdomains and/or protein domains will have different starting pIs whichare exploited in the present invention. In general, as outlined herein,the pIs are engineered to result in a total pI difference of eachmonomer of at least about 0.1 logs, with 0.2 to 0.5 being preferred asoutlined herein.

Furthermore, as will be appreciated by those in the art and outlinedherein, in some embodiments, heterodimers can be separated fromhomodimers on the basis of size. As shown in the Figures, for example,several of the formats allow separation of heterodimers and homodimerson the basis of size.

In the case where pI variants are used to achieve heterodimerization, byusing the constant region(s) of Fc domains(s), a more modular approachto designing and purifying heterodimeric Fc fusion proteins is provided.Thus, in some embodiments, heterodimerization variants (including skewand purification heterodimerization variants) must be engineered. Inaddition, in some embodiments, the possibility of immunogenicityresulting from the pI variants is significantly reduced by importing pIvariants from different IgG isotypes such that pI is changed withoutintroducing significant immunogenicity. Thus, an additional problem tobe solved is the elucidation of low pI constant domains with high humansequence content, e.g. the minimization or avoidance of non-humanresidues at any particular position.

A side benefit that can occur with this pI engineering is also theextension of serum half-life and increased FcRn binding. That is, asdescribed in U.S. Ser. No. 13/194,904 (incorporated by reference in itsentirety), lowering the pI of antibody constant domains (including thosefound in antibodies and Fc fusions) can lead to longer serum retentionin vivo. These pI variants for increased serum half life also facilitatepI changes for purification.

In addition, it should be noted that the pI variants of theheterodimerization variants give an additional benefit for the analyticsand quality control process of Fc fusion proteins, as the ability toeither eliminate, minimize and distinguish when homodimers are presentis significant. Similarly, the ability to reliably test thereproducibility of the heterodimeric Fc fusion protein production isimportant.

A. Heterodimerization Variants

The present invention provides heterodimeric proteins, includingheterodimeric Fc fusion proteins in a variety of formats, which utilizeheterodimeric variants to allow for heterodimeric formation and/orpurification away from homodimers. The heterodimeric fusion constructsare based on the self-assembling nature of the two Fc domains, e.g., two“monomers” that assemble into a “dimer”. **Robin

There are a number of suitable pairs of sets of heterodimerization skewvariants. These variants come in “pairs” of “sets”. That is, one set ofthe pair is incorporated into the first monomer and the other set of thepair is incorporated into the second monomer. It should be noted thatthese sets do not necessarily behave as “knobs in holes” variants, witha one-to-one correspondence between a residue on one monomer and aresidue on the other; that is, these pairs of sets form an interfacebetween the two monomers that encourages heterodimer formation anddiscourages homodimer formation, allowing the percentage of heterodimersthat spontaneously form under biological conditions to be over 90%,rather than the expected 50% (25% homodimer A/A:50% heterodimer A/B:25%homodimer B/B).

B. Steric Variants

In some embodiments, the formation of heterodimers can be facilitated bythe addition of steric variants. That is, by changing amino acids ineach heavy chain, different heavy chains are more likely to associate toform the heterodimeric structure than to form homodimers with the sameFc amino acid sequences. Suitable steric variants are included in in theFIG. 29 of U.S. Ser. No. 15/141,350, all of which is hereby incorporatedby reference in its entirety, as well as in FIGS. 1A-1E.

One mechanism is generally referred to in the art as “knobs and holes”,referring to amino acid engineering that creates steric influences tofavor heterodimeric formation and disfavor homodimeric formation canalso optionally be used; this is sometimes referred to as “knobs andholes”, as described in U.S. Ser. No. 61/596,846, Ridgway et al.,Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997270:26; U.S. Pat. No. 8,216,805, all of which are hereby incorporated byreference in their entirety. The Figures identify a number of “monomerA-monomer B” pairs that rely on “knobs and holes”. In addition, asdescribed in Merchant et al., Nature Biotech. 16:677 (1998), these“knobs and hole” mutations can be combined with disulfide bonds to skewformation to heterodimerization.

An additional mechanism that finds use in the generation of heterodimersis sometimes referred to as “electrostatic steering” as described inGunasekaran et al., J. Biol. Chem. 285(25): 19637 (2010), herebyincorporated by reference in its entirety. This is sometimes referred toherein as “charge pairs”. In this embodiment, electrostatics are used toskew the formation towards heterodimerization. As those in the art willappreciate, these may also have have an effect on pI, and thus onpurification, and thus could in some cases also be considered pIvariants. However, as these were generated to force heterodimerizationand were not used as purification tools, they are classified as “stericvariants”. These include, but are not limited to, D221E/P228E/L368Epaired with D221R/P228R/K409R (e.g., these are “monomer correspondingsets) and C220E/P228E/368E paired with C220R/E224R/P228R/K409R.

Additional monomer A and monomer B variants that can be combined withother variants, optionally and independently in any amount, such as pIvariants outlined herein or other steric variants that are shown in FIG.37 of US 2012/0149876, all of which are incorporated expressly byreference herein.

In some embodiments, the steric variants outlined herein can beoptionally and independently incorporated with any pI variant (or othervariants such as Fc variants, FcRn variants, etc.) into one or bothmonomers, and can be independently and optionally included or excludedfrom the proteins of the invention.

A list of suitable skew variants is found in FIG. 3 . Of particular usein many embodiments are the pairs of sets including, but not limited to,S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L, K370S:S364K/E357Q andT366S/L368A/Y407V:T366W (optionally including a bridging disulfide,T366S/L368A/Y407V/Y349C:T366W/S354C). In terms of nomenclature, the pair“S364K/E357Q:L368D/K370S” means that one of the monomers has the doublevariant set S364K/E357Q and the other has the double variant setL368D/K370S; as above, the “strandedness” of these pairs depends on thestarting pI.

C. pI (Isoelectric Point) Variants for Heterodimers

In general, as will be appreciated by those in the art, there are twogeneral categories of pI variants: those that increase the pI of theprotein (basic changes) and those that decrease the pI of the protein(acidic changes). As described herein, all combinations of thesevariants can be done: one monomer may be wild type, or a variant thatdoes not display a significantly different pI from wild-type, and theother can be either more basic or more acidic. Alternatively, eachmonomer is changed, one to more basic and one to more acidic.

Preferred combinations of pI variants are shown in FIG. 30 of U.S. Ser.No. 15/141,350, all of which are herein incorporated by reference in itsentirety. As outlined herein and shown in the figures, these changes areshown relative to IgG1, but all isotypes can be altered this way, aswell as isotype hybrids. In the case where the heavy chain constantdomain is from IgG2-4, R133E and R133Q can also be used.

In one embodiment, a preferred combination of pI variants has onemonomer comprising 208D/295E/384D/418E/421D variants(N208D/Q295E/N384D/Q418E/N421D when relative to human IgG1) if one ofthe Fc monomers includes a CH1 domain. In some instances, the secondmonomer comprising a positively charged domain linker, including(GKPGS)₄ (SEQ ID NO: 5). In some cases, the first monomer includes a CH1domain, including position 208. Accordingly, in constructs that do notinclude a CH1 domain (for example for heterodimeric Fc fusion proteinsthat do not utilize a CH1 domain on one of the domains), a preferrednegative pI variant Fc set includes 295E/384D/418E/421D variants(Q295E/N384D/Q418E/N421D when relative to human IgG1).

In some embodiments, mutations are made in the hinge domain of the Fcdomain, including positions 221, 222, 223, 224, 225, 233, 234, 235 and236. It should be noted that changes in 233-236 can be made to increaseeffector function (along with 327A) in the IgG2 backbone. Thus, pImutations and particularly substitutions can be made in one or more ofpositions 221-225, with 1, 2, 3, 4 or 5 mutations finding use in thepresent invention. Again, all possible combinations are contemplated,alone or with other pI variants in other domains.

Specific substitutions that find use in lowering the pI of hinge domainsinclude, but are not limited to, a deletion at position 221, anon-native valine or threonine at position 222, a deletion at position223, a non-native glutamic acid at position 224, a deletion at position225, a deletion at position 235 and a deletion or a non-native alanineat position 236. In some cases, only pI substitutions are done in thehinge domain, and in others, these substitution(s) are added to other pIvariants in other domains in any combination.

In some embodiments, mutations can be made in the CH2 region, includingpositions 274, 296, 300, 309, 320, 322, 326, 327, 334 and 339. Again,all possible combinations of these 10 positions can be made; e.g., a pIantibody may have 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 CH2 pI substitutions.

Specific substitutions that find use in lowering the pI of CH2 domainsinclude, but are not limited to, a non-native glutamine or glutamic acidat position 274, a non-native phenylalanine at position 296, a nonnative phenylalanine at position 300, a non-native valine at position309, a non-native glutamic acid at position 320, a non-native glutamicacid at position 322, a non-native glutamic acid at position 326, anon-native glycine at position 327, a non-native glutamic acid atposition 334, a non native threonine at position 339, and all possiblecombinations within CH2 and with other domains.

In this embodiment, the mutations can be independently and optionallyselected from position 355, 359, 362, 384, 389, 392, 397, 418, 419, 444and 447. Specific substitutions that find use in lowering the pI of CH3domains include, but are not limited to, a non native glutamine orglutamic acid at position 355, a non-native serine at position 384, anon-native asparagine or glutamic acid at position 392, a non-nativemethionine at position 397, a non native glutamic acid at position 419,a non native glutamic acid at position 359, a non native glutamic acidat position 362, a non native glutamic acid at position 389, a nonnative glutamic acid at position 418, a non native glutamic acid atposition 444, and a deletion or non-native aspartic acid at position447. Exemplary embodiments of pI variants are provided in FIG. 2 .

D. Isotypic Variants

In addition, many embodiments of the invention rely on the “importation”of pI amino acids at particular positions from one IgG isotype intoanother, thus reducing or eliminating the possibility of unwantedimmunogenicity being introduced into the variants. A number of these areshown in FIG. 21 of US Publ. App. No. 2014/0370013, hereby incorporatedby reference. That is, IgG1 is a common isotype for therapeuticantibodies for a variety of reasons, including high effector function.However, the heavy constant region of IgG1 has a higher pI than that ofIgG2 (8.10 versus 7.31). By introducing IgG2 residues at particularpositions into the IgG1 backbone, the pI of the resulting monomer islowered (or increased) and additionally exhibits longer serum half-life.For example, IgG1 has a glycine (pI 5.97) at position 137, and IgG2 hasa glutamic acid (pI 3.22); importing the glutamic acid will affect thepI of the resulting protein. As is described below, a number of aminoacid substitutions are generally required to significant affect the pIof the variant Fc fusion protein. However, it should be noted asdiscussed below that even changes in IgG2 molecules allow for increasedserum half-life.

In other embodiments, non-isotypic amino acid changes are made, eitherto reduce the overall charge state of the resulting protein (e.g., bychanging a higher pI amino acid to a lower pI amino acid), or to allowaccommodations in structure for stability, etc. as is more furtherdescribed below.

In addition, by pI engineering both the heavy and light constantdomains, significant changes in each monomer of the heterodimer can beseen. As discussed herein, having the pIs of the two monomers differ byat least 0.5 can allow separation by ion exchange chromatography orisoelectric focusing, or other methods sensitive to isoelectric point.

E. Calculating pI

The pI of each monomer can depend on the pI of the variant heavy chainconstant domain and the pI of the total monomer, including the variantheavy chain constant domain and the fusion partner. Thus, in someembodiments, the change in pI is calculated on the basis of the variantheavy chain constant domain, using the chart in the FIG. 19 of US Publ.App. No. 2014/0370013. As discussed herein, which monomer to engineer isgenerally decided by the inherent pI of each monomer.

F. pI Variants that Also Confer Better FcRn In Vivo Binding

In the case where the pI variant decreases the pI of the monomer, theycan have the added benefit of improving serum retention in vivo.

Although still under examination, Fc regions are believed to have longerhalf-lives in vivo, because binding to FcRn at pH 6 in an endosomesequesters the Fc (Ghetie and Ward, 1997 Immunol Today. 18(12): 592-598,entirely incorporated by reference). The endosomal compartment thenrecycles the Fc to the cell surface. Once the compartment opens to theextracellular space, the higher pH, ˜7.4, induces the release of Fc backinto the blood. In mice, Dall' Acqua et al. showed that Fc mutants withincreased FcRn binding at pH 6 and pH 7.4 actually had reduced serumconcentrations and the same half-life as wild-type Fc (Dall' Acqua etal. 2002, J. Immunol. 169:5171-5180, entirely incorporated byreference). The increased affinity of Fc for FcRn at pH 7.4 is thoughtto forbid the release of the Fc back into the blood. Therefore, the Fcmutations that will increase Fc's half-life in vivo will ideallyincrease FcRn binding at the lower pH while still allowing release of Fcat higher pH. The amino acid histidine changes its charge state in thepH range of 6.0 to 7.4. Therefore, it is not surprising to find Hisresidues at important positions in the Fc/FcRn complex.

G. Additional Fc Variants for Additional Functionality

In addition to pI amino acid variants, there are a number of useful Fcamino acid modification that can be made for a variety of reasons,including, but not limited to, altering binding to one or more FcγRreceptors, altered binding to FcRn receptors, etc.

Accordingly, the proteins of the invention can include amino acidmodifications, including the heterodimerization variants outlinedherein, which includes the pI variants and steric variants. Each set ofvariants can be independently and optionally included or excluded fromany particular heterodimeric protein.

H. FcγR Variants

Accordingly, there are a number of useful Fc substitutions that can bemade to alter binding to one or more of the FcγR receptors.Substitutions that result in increased binding as well as decreasedbinding can be useful. For example, it is known that increased bindingto FcγRIIIa results in increased ADCC (antibody dependent cell-mediatedcytotoxicity; the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell). Similarly, decreasedbinding to FcγRIIb (an inhibitory receptor) can be beneficial as well insome circumstances. Amino acid substitutions that find use in thepresent invention include those listed in U.S. Ser. No. 11/124,620(particularly FIG. 41 ), Ser. Nos. 11/174,287, 11/396,495, 11/538,406,all of which are expressly incorporated herein by reference in theirentirety and specifically for the variants disclosed therein. Particularvariants that find use include, but are not limited to, 236A, 239D,239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E,239D/332E/330Y, 239D, 332E/330L, 243A, 243L, 264A, 264V and 299T.

In addition, amino acid substitutions that increase affinity for FcγRIIccan also be included in the Fc domain variants outlined herein. Thesubstitutions described in, for example, U.S. Ser. Nos. 11/124,620 and14/578,305 are useful.

In addition, there are additional Fc substitutions that find use inincreased binding to the FcRn receptor and increased serum half-life, asspecifically disclosed in U.S. Ser. No. 12/341,769, hereby incorporatedby reference in its entirety, including, but not limited to, 434S, 434A,428L, 308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S,436V/428L and 259I/308F/428L.

I. Ablation Variants

Similarly, another category of functional variants are “FcγR ablationvariants” or “Fc knock out (FcKO or KO)” variants. In these embodiments,for some therapeutic applications, it is desirable to reduce or removethe normal binding of the Fc domain to one or more or all of the Fcγreceptors (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) to avoidadditional mechanisms of action. That is, for example, in manyembodiments, particularly in the use of bispecific immunomodulatoryantibodies desirable to ablate FcγRIIIa binding to eliminate orsignificantly reduce ADCC activity such that one of the Fc domainscomprises one or more Fcγ receptor ablation variants. These ablationvariants are depicted in FIG. 31 of U.S. Ser. No. 15/141,350, all ofwhich are herein incorporated by reference in its entirety, and each canbe independently and optionally included or excluded, with preferredaspects utilizing ablation variants selected from the group consistingof G236R/L328R, E233P/L234V/L235A/G236del/S239K,E233P/L234V/L235A/G236del/S267K, E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G and E233P/L234V/L235A/G236del,according to the EU index. It should be noted that the ablation variantsreferenced herein ablate FcγR binding but generally not FcRn binding.

Exemplary embodiments of pI variants are provided in FIG. 3 .

J. Combination of Heterodimeric and Fc Variants

As will be appreciated by those in the art, all of the recitedheterodimerization variants (including skew and/or pI variants) can beoptionally and independently combined in any way, as long as they retaintheir “strandedness” or “monomer partition”. In addition, all of thesevariants can be combined into any of the heterodimerization formats.

In the case of pI variants, while embodiments finding particular use areshown in the Figures, other combinations can be generated, following thebasic rule of altering the pI difference between two monomers tofacilitate purification.

In addition, any of the heterodimerization variants, skew and pI, arealso independently and optionally combined with Fc ablation variants, Fcvariants, FcRn variants, as generally outlined herein.

In addition, a monomeric Fc domain can comprise a set of amino acidsubstitutions that includes C220S/S267K/L368D/K370S orC220S/S267K/S364K/E357Q.

In addition, the heterodimeric Fc fusion proteins can comprise skewvariants (e.g., a set of amino acid substitutions as shown in FIGS.1A-1C of U.S. Ser. No. 15/141,350, all of which are herein incorporatedby reference in its entirety), with particularly useful skew variantsbeing selected from the group consisting of S364K/E357Q:L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411T/E360E/Q362E:D401K;L368D/K370S:S364K/E357L, K370S:S364K/E357Q, T366S/L368A/Y407V:T366W andT366S/L368A/Y407V/Y349C:T366W/S354C, optionally ablation variants,optionally charged domain linkers and the heavy chain comprises pIvariants.

In some embodiments, the Fc domain comprising an amino acid substitutionselected from the group consisting of: 236R, 239D, 239E, 243L, M252Y,V259I, 267D, 267E, 298A, V308F, 328F, 328R, 330L, 332D, 332E, M428L,N434A, N434S, 236R/328R, 239D/332E, M428L, 236R/328F, V259I/V308F,267E/328F, M428L/N434S, Y436I/M428L, Y436V/M428L, Y436I/N434S,Y436V/N434S, 239D/332E/330L, M252Y/S254T/T256E, V259I/V308F/M428L,E233P/L234V/L235A/G236del/S267K, G236R/L328R and PVA/S267K. In somecases, the Fc domain comprises the amino acid substitution 239D/332E. Inother cases, the Fc domain comprises the amino acid substitutionG236R/L328R or PVA/S267K.

In one embodiment, a particular combination of skew and pI variants thatfinds use in the present invention is T366S/L368A/Y407V:T366W(optionally including a bridging disulfide,T366S/L368A/Y407V/Y349C:T366W/S354C) with one monomer comprisesQ295E/N384D/Q418E/N481D and the other a positively charged domainlinker. As will be appreciated in the art, the “knobs in holes” variantsdo not change pI, and thus can be used on either monomer.

IV. Useful Formats of the Invention

As shown in FIG. 64 , there are a number of useful formats of thebispecific heterodimeric fusion proteins of the invention. In general,the heterodimeric fusion proteins of the invention have three functionalcomponents: an IL-15/IL-15Rα(sushi) component, an anti-PD-1 component,and an Fc component, each of which can take different forms as outlinedherein and each of which can be combined with the other components inany configuration.

The first and the second Fc domains can have a set of amino acidsubstitutions selected from the group consisting of a)S267K/L368D/K370S:S267K/LS364K/E357Q; b) S364K/E357Q:L368D/K370S; c)L368D/K370S:S364K; d) L368E/K370S:S364K; e) T411T/E360E/Q362E:D401K; f)L368D/K370S:S364K/E357L and g) K370S:S364K/E357Q, according to EUnumbering.

In some embodiments, the first and/or the second Fc domains have anadditional set of amino acid substitutions comprisingQ295E/N384D/Q418E/N421D, according to EU numbering.

Optionally, the first and/or the second Fc domains have an additionalset of amino acid substitutions consisting of G236R/L328R,E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K,E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G and E233P/L234V/L235A/G236del,according to EU numbering.

Optionally, the first and/or second Fc domains have 428L/434S variantsfor half life extension.

A. scIL-15/Rα×scFv

One embodiment is shown in FIG. 64A, and comprises two monomers. Thefirst monomer comprises, from N- to C-terminus, the sushi domain-domainlinker-Il-15-domain linker-CH2-CH3, and the second monomer comprisesvh-scFv linker-vl-hinge-CH2-CH3 or vl-scFv linker-vh-hinge-CH2-CH3,although in either orientation a domain linker can be substituted forthe hinge. This is generally referred to as “scIL-15/Rα×scFv”, with the“sc” standing for “single chain” referring to the attachment of theIL-15 and suchi domain using a covalent linker. Preferred combinationsof variants for this embodiment are found in FIGS. 7A and B.

In the scIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14.

In the scIL-15/Rα×scFv format, one preferred embodiment utilizes theskew variant pair S364K/E357Q:L368D/K370S.

In the scIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14and the skew variant pair S364K/E357Q:L368D/K370S.

In the scIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14, in the FIG. 7A format: e.g. the skew variants S364K/E357Q (on thescFv-Fc monomer) and L368D/K370S (on the IL-15 complex monomer), the pIvariants Q295E/N384D/Q418E/N421D (on the IL-15 complex side), theablation variants E233P/L234V/L235A/G236_/S267K on both monomers, andoptionally the 428L/434S variants on both sides.

In the scIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14, in the FIG. 7B format.

In the scIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having the variable heavy and variable light sequencesfrom 1C11[PD-1]_H3L3 as shown in FIG. 14 .

In the scIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 inthe FIG. 7A format.

In the scIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 inthe FIG. 7B format.

In the scIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having an Fv sequence as depicted in FIG. 86 .

In the scIL-15/Rα×scFv format, one preferred embodiment utilizes theIL-15 complex (sushi domain-linker-IL-15) as depicted in FIG. 87A.

In the scIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14and the IL-15 complex (sushi domain-linker-IL-15) as depicted in FIG.87A.

B. scFv×ncIL-15/Rα

This embodiment is shown in FIG. 64B, and comprises three monomers. Thefirst monomer comprises, from N- to C-terminus, the sushi domain-domainlinker-CH2-CH3, and the second monomer comprises vh-scFvlinker-vl-hinge-CH2-CH3 or vl-scFv linker-vh-hinge-CH2-CH3, although ineither orientation a domain linker can be substituted for the hinge. Thethird monomer is the IL-15 domain. This is generally referred to as“ncIL-15/Rα×scFv” or “scFv×ncIL-15/Rα” with the “nc” standing for“non-covalent” referring to the self-assembling non-covalent attachmentof the IL-15 and suchi domain. Preferred combinations of variants forthis embodiment are found in FIGS. 7A and B.

In the ncIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14.

In the ncIL-15/Rα×scFv format, one preferred embodiment utilizes theskew variant pair S364K/E357Q:L368D/K370S.

In the ncIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14and the skew variant pair S364K/E357Q:L368D/K370S.

In the ncIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14, in the FIG. 7A format: e.g. the skew variants S364K/E357Q (on thescFv-Fc monomer) and L368D/K370S (on the IL-15 complex monomer), the pIvariants Q295E/N384D/Q418E/N421D (on the IL-15 complex side), theablation variants E233P/L234V/L235A/G236_/S267K on both monomers, andoptionally the 428L/434S variants on both sides.

In the ncIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14, in the FIG. 7B format.

In the ncIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having the variable heavy and light sequences from1C11[PD-1]_H3L3 as shown in FIG. 14 .

In the ncIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 inthe FIG. 7A format.

In the ncIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 inthe FIG. 7B format.

In the ncIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having an Fv sequence as depicted in FIG. 86 .

In the scIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14and the IL-15 complex (sushi domain and IL-15) as depicted in FIG. 87B.

C. scFv×dsIL-15/Rα

This embodiment is shown in FIG. 64C, and comprises three monomers. Thefirst monomer comprises, from N- to C-terminus, the sushi domain-domainlinker-CH2-CH3, wherein the sushi domain has an engineered cysteineresidue and the second monomer comprises vh-scFv linker-vl-hinge-CH2-CH3or vl-scFv linker-vh-hinge-CH2-CH3, although in either orientation adomain linker can be substituted for the hinge. The third monomer is theIL-15 domain, also engineered to have a cysteine variant amino acid,thus allowing a disulfide bridge to form between the sushi domain andthe IL-15 domain. This is generally referred to as “scFv×dsIL-15/Rα” ordsIL-15/Rα×scFv, with the “ds” standing for “disulfide”. Preferredcombinations of variants for this embodiment are found in FIGS. 7A andB.

In the dsIL-15/Rα×scFv format, one preferred embodiment utilizes theskew variant pair S364K/E357Q:L368D/K370S.

In the dsIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14.

In the dsIL-15/Rα×scFv format, one preferred embodiment utilizes theskew variant pair S364K/E357Q:L368D/K370S and the anti-PD-1 ABD havingthe sequence 1G6_L1.194_H1.279 as shown in FIG. 14 .

In the dsIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14, in the FIG. 7A format: e.g. the skew variants S364K/E357Q (on thescFv-Fc monomer) and L368D/K370S (on the IL-15 complex monomer), the pIvariants Q295E/N384D/Q418E/N421D (on the IL-15 complex side), theablation variants E233P/L234V/L235A/G236_/S267K on both monomers, andoptionally the 428L/434S variants on both sides.

In the dsIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14, in the FIG. 7B format.

In the dsIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 .

In the dsIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 inthe FIG. 7A format.

In the dsIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 inthe FIG. 7B format.

In the dsIL-15/Rα×scFv format, one preferred embodiment utilizes theanti-PD-1 ABD having an Fv sequence as depicted in FIG. 86 .

D. scIL-15/Rα×Fab

This embodiment is shown in FIG. 64D, and comprises three monomers.

The first monomer comprises, from N- to C-terminus, the sushidomain-domain linker-IL-15-domain linker-CH2-CH3 and the second monomercomprises a heavy chain, VH-CH1-hinge-CH2-CH3. The third monomer is alight chain, VL-CL. This is generally referred to as “scIL-15/Rα×Fab”,with the “sc” standing for “single chain”. Preferred combinations ofvariants for this embodiment are found in FIG. 7C.

In the scIL-15/Rα×Fab format, one preferred embodiment utilizes the skewvariant pair S364K/E357Q:L368D/K370S.

In the scIL-15/Rα×Fab format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14.

In the scIL-15/Rα×Fab format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14, in the FIG. 7A format: e.g. the skew variants S364K/E357Q (on thescFv-Fc monomer) and L368D/K370S (on the IL-15 complex monomer), the pIvariants Q295E/N384D/Q418E/N421D (on the IL-15 complex side), theablation variants E233P/L234V/L235A/G236_/S267K on both monomers, andoptionally the 428L/434S variants on both sides.

In the scIL-15/Rα×Fab format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14, in the FIG. 7C format.

In the scIL-15/Rα×Fab format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14and the skew variant pair S364K/E357Q:L368D/K370S.

In the scIL-15/Rα×Fab format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 .

In the scIL-15/Rα×Fab format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 inthe FIG. 7C format.

In the scIL-15/Rα×Fab format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 inthe FIG. 7C format.

In the scIL-15/Rα×Fab format, one preferred embodiment utilizes theanti-PD-1 ABD having an Fv sequence as depicted in FIG. 86 .

In the scIL-15/Rα×Fab format, one preferred embodiment utilizes theIL-15 complex sequences depicted in FIG. 87A.

In the scIL-15/Rα×Fab format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14and the IL-15 complex (sushi domain-linker-IL-15) as depicted in FIG.87A.

E. Fab×ncIL-15/Rα

This embodiment is shown in FIG. 64E, and comprises three monomers. Thefirst monomer comprises, from N- to C-terminus, the sushi domain-domainlinker-CH2-CH3, and the second monomer comprises a heavy chain,VH-CH1-hinge-CH2-CH3. The third monomer is the IL-15 domain. This isgenerally referred to as “Fab×ncIL-15/Rα”, with the “nc” standing for“non-covalent” referring to the self-assembling non-covalent attachmentof the IL-15 and suchi domain. Preferred combinations of variants forthis embodiment are found in FIG. 7D.

In the Fab×ncIL-15/Rα format, one preferred embodiment utilizes the skewvariant pair S364K/E357Q:L368D/K370S.

In the Fab×ncIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14.

In the Fab×ncIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14, in the FIG. 7D format.

In the Fab×ncIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14, in the FIG. 7D format.

In the Fab×ncIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14and the skew variant pair S364K/E357Q:L368D/K370S.

In the Fab×ncIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 inthe FIG. 7D format.

In the Fab×ncIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 inthe FIG. 7D format.

In the Fab×ncIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having an Fv sequence as depicted in FIG. 86 .

F. Fab×dsIL-15/Rα

This embodiment is shown in FIG. 64F, and comprises three monomers. Thefirst monomer comprises, from N- to C-terminus, the sushi domain-domainlinker-CH2-CH3, wherein the sushi domain has been engineered to containa cysteine residue, and the second monomer comprises a heavy chain,VH-CH1-hinge-CH2-CH3. The third monomer is the IL-15 domain, alsoengineered to have a cysteine residue, such that a disulfide bridge isformed under native cellular conditions. This is generally referred toas “Fab×dsIL-15/Rα”, with the “ds” standing for “disulfide” referring tothe self-assembling non-covalent attachment of the IL-15 and suchidomain. Preferred combinations of variants for this embodiment are foundin FIG. 7 .

In the dsIL-15/Rα×Fab format, one preferred embodiment utilizes the skewvariant pair S364K/E357Q:L368D/K370S.

In the Fab×dsIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14.

In the Fab×dsIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14, in the FIG. 7D format.

In the Fab×dsIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14, in the FIG. 7D format.

In the Fab×dsIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 .

In the Fab×dsIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14and the skew variant pair S364K/E357Q:L368D/K370S.

In the Fab×dsIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 inthe FIG. 7D format.

In the Fab×dsIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 inthe FIG. 7D format.

In the Fab×dsIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having an Fv sequence as depicted in FIG. 86 .

In the Fab×dsIL-15/Rα format, one preferred embodiment utilizes theIL-15 complex sequences depicted in FIG. 87B.

G. mAb-scIL-15/Rα

This embodiment is shown in FIG. 64G, and comprises three monomers(although the fusion protein is a tetramer). The first monomer comprisesa heavy chain, VH-CH1-hinge-CH2-CH3. The second monomer comprises aheavy chain with a scIL-15 complex, VH-CH1-hinge-CH2-CH3-domainlinker-sushi domain-domain linker-IL-15. The third (and fourth) monomerare light chains, VL-CL. This is generally referred to as“mAb-scIL-15/Rα”, with the “sc” standing for “single chain”. Preferredcombinations of variants for this embodiment are found in FIG. 7 .

In the mAb-scIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14.

In the mAb-scIL-15/Rα format, one preferred embodiment utilizes the skewvariant pair S364K/E357Q:L368D/K370S.

In the mAb-scIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14, in the FIG. 7A format.

In the mAb-scIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14, in the FIG. 7C format.

In the mAb-scIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 .

In the mAb-scIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14and the skew variant pair S364K/E357Q:L368D/K370S.

In the mAb-scIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 inthe FIG. 7C format.

In the mAb-scIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 inthe FIG. 7C format.

In the mAb-scIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having an Fv sequence as depicted in FIG. 86 .

In the mAb-scIL-15/Rα format, one preferred embodiment utilizes theIL-15 complex sequences depicted in FIG. 87A.

H. mAb-ncIL-15/Rα

This embodiment is shown in FIG. 64H, and comprises four monomers(although the heterodimeric fusion protein is a pentamer). The firstmonomer comprises a heavy chain, VH-CH1-hinge-CH2-CH3. The secondmonomer comprises a heavy chain with an IL-15Rα(sushi) domain,VH-CH1-hinge-CH2-CH3-domain linker-sushi domain. The third monomer is anIL-15 domain. The fourth (and fifth) monomer are light chains, VL-CL.This is generally referred to as “mAb-ncIL-15/Rα”, with the “nc”standing for “non-covalent”. Preferred combinations of variants for thisembodiment are found in FIG. 7 .

In the mAb-ncIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14.

In the mAb-ncIL-15/Rα format, one preferred embodiment utilizes the skewvariant pair S364K/E357Q:L368D/K370S.

In the mAb-ncIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14, in the FIG. 7 format.

In the mAb-ncIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14, in the FIG. 7 format.

In the mAb-ncIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 .

In the mAb-ncIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14and the skew variant pair S364K/E357Q:L368D/K370S.

In the mAb-ncIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 inthe FIG. 7 format.

In the mAb-ncIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 inthe FIG. 7 format.

In the mAb-ncIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having an Fv sequence as depicted in FIG. 86 .

I. mAb-dsIL-15/Rα

This embodiment is shown in FIG. 64I, and comprises four monomers(although the heterodimeric fusion protein is a pentamer). The firstmonomer comprises a heavy chain, VH-CH1-hinge-CH2-CH3. The secondmonomer comprises a heavy chain with an IL-15Rα(sushi) domain:VH-CH1-hinge-CH2-CH3-domain linker-sushi domain, where the sushi domainhas been engineered to contain a cysteine residue. The third monomer isan IL-15 domain, which has been engineered to contain a cysteineresidue, such that the IL-15 complex is formed under physiologicalconditions. The fourth (and fifth) monomer are light chains, VL-CL. Thisis generally referred to as “mAb-ncIL-15/Rα”, with the “nc” standing for“non-covalent”. Preferred combinations of variants for this embodimentare found in FIG. 7 .

In the mAb-dsIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14.

In the mAb-dsIL-15/Rα format, one preferred embodiment utilizes the skewvariant pair S364K/E357Q:L368D/K370S.

In the mAb-dsIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14, in the FIG. 7 format.

In the mAb-dsIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14, in the FIG. 7 format.

In the mAb-ncIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 .

In the mAb-dsIL-15/Rα format, one preferred embodiment utilizes the skewvariant pair S364K/E357Q:L368D/K370S and utilizes the anti-PD-1 ABDhaving the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 .

In the mAb-dsIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 inthe FIG. 7 format.

In the mAb-dsIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 inthe FIG. 7 format.

In the mAb-dsIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having an Fv sequence as depicted in FIG. 86 .

J. Central-IL-15/Rα

This embodiment is shown in FIG. 64J, and comprises four monomersforming a tetramer. The first monomer comprises a VH-CH1-[optionaldomain linker]-IL-15-[optional domain linker]-CH2-CH3, with the secondoptional domain linker sometimes being the hinge domain. The secondmonomer comprises a VH-CH1-[optional domain linker]-sushidomain-[optional domain linker]-CH2-CH3, with the second optional domainlinker sometimes being the hinge domain. The third (and fourth) monomersare light chains, VL-CL. This is generally referred to as“Central-IL-15/Rα”. Preferred combinations of variants for thisembodiment are found in FIG. 7 .

In the Central-IL-15/Rα format, one preferred embodiment utilizes theskew variant pair S364K/E357Q:L368D/K370S

In the Central-IL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14.

In the Central-IL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14, in the FIG. 7 format.

In the Central-IL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14, in the FIG. 7 format.

In the Central-IL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 .

In the Central-IL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14and the skew variant pair S364K/E357Q:L368D/K370S.

In the Central-IL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 inthe FIG. 7 format.

In the Central-IL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 inthe FIG. 7 format.

In the Central-IL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having an Fv sequence as depicted in FIG. 86 .

K. Central scIL-15/Rα

This embodiment is shown in FIG. 64K, and comprises four monomersforming a tetramer. The first monomer comprises a VH-CH1-[optionaldomain linker]-sushi domain-domain linker-IL-15-[optional domainlinker]-CH2-CH3, with the second optional domain linker sometimes beingthe hinge domain. The second monomer comprises a VH-CH1-hinge-CH2-CH3.The third (and fourth) monomers are light chains, VL-CL. This isgenerally referred to as “Central-scIL-15/Rα”, with the “sc” standingfor “single chain”.

Preferred combinations of variants for this embodiment are found in FIG.7 .

In the Central-scIL-15/Rα format, one preferred embodiment utilizes theskew variant pair S364K/E357Q:L368D/K370S.

In the Central-scIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14.

In the Central-scIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14, in the FIG. 7 format.

In the Central-scIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1G6_L1.194_H1.279 as shown in FIG. 14, in the FIG. 7 format.

In the Central-scIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 .

In the Central-scIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14and the skew variant pair S364K/E357Q:L368D/K370S.

In the Central-scIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 inthe FIG. 7 format.

In the Central-scIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having the sequence 1C11[PD-1]_H3L3 as shown in FIG. 14 inthe FIG. 7 format.

In the Central-scIL-15/Rα format, one preferred embodiment utilizes theanti-PD-1 ABD having an Fv sequence as depicted in FIG. 86 .

In the Central-scIL-15/Rα format, one preferred embodiment utilizes thescIL-15 complex sequence of FIG. 87A.

V. IL-15/IL-15Rα-Fc Fusion Monomers

The bispecific heterodimeric fusion proteins of the present inventioninclude an IL-15/IL-15 receptor alpha (IL-15Rα)-Fc fusion monomer;reference is made to U.S. application entitled “IL15/IL15RαHETERODIMERIC FC-FUSION PROTEINS”, filed concurrently herewith on 16Oct. 2017,” and U.S. Ser. No. 62/408,655, filed on Oct. 14, 2016, U.S.Ser. No. 62/443,465, filed on Jan. 6, 2017, and U.S. Ser. No.62/477,926, filed on Mar. 28, 2017, hereby incorporated by reference intheir entirety and in particular for the sequences outlined therein. Insome cases, the IL-15 and IL-15 receptor alpha (IL-15Rα) protein domainsare in different orientations. Exemplary embodiments of IL-15/IL-15Rα-Fcfusion monomers are provided in XENP21480 (chain 1; FIG. 64A), XENP22022(chain 1, FIG. 64D), XENP22112, (chains 1 and 3; FIG. 64E), XENP22641(chains 2 and 4; FIG. 64F), XENP22642, (chains 1 and 4; FIG. 64H) andXENP22644 (chains 1 and 4; FIG. 64I).

In some embodiments, the human IL-15 protein has the amino acid sequenceset forth in NCBI Ref. Seq. No. NP_000576.1 or SEQ ID NO: 1. In somecases, the coding sequence of human IL-15 is set forth in NCBI Ref. Seq.No. NM_000585. An exemplary IL-15 protein of the Fc fusion heterodimericprotein outlined herein can have the amino acid sequence of SEQ ID NO:2or amino acids 49-162 of SEQ ID NO: 1. In some embodiments, the IL-15protein has at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to SEQ ID NO:2. In some embodiments,the IL-15 protein has the amino acid sequence set forth in SEQ ID NO:2and the amino acid substitution N72D. In other embodiments, the IL-15protein has the amino acid sequence of SEQ ID NO:2 and one or more aminoacid substitutions selected from the group consisting of C42S, L45C,Q48C, V49C, L52C, E53C, E87C, and E89C. In some aspects, the IL15protein has one or more amino acid substitutions selected from the groupconsisting of N1D, N4D, D8N, D30N, D61N, E64Q, N65D, and Q108E. In otherembodiments, the amino acid substitutions are N4D/N65D. In someembodiments, the amino acid substitution is Q108E. In certainembodiments, the amino acid substitution is N65D. In other embodiments,the amino acid substitutions are D30N/E64Q/N65D. In certain embodiments,the amino acid substitution is N65D. In some instances, the amino acidsubstitutions are N1D/N65D. Optionally, the IL-15 protein also has anN72D substitution. The IL-15 protein of the Fc fusion protein can have1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions.

In some embodiments, the human IL-15 receptor alpha (IL-15Rα) proteinhas the amino acid sequence set forth in NCBI Ref. Seq. No. NP_002180.1or SEQ ID NO:3. In some cases, the coding sequence of human IL-15Rα isset forth in NCBI Ref. Seq. No. NM_002189.3. An exemplary the IL-15Rαprotein of the Fc fusion heterodimeric protein outlined herein cancomprise or consist of the sushi domain of SEQ ID NO:3 (e.g., aminoacids 31-95 of SEQ ID NO:3), or in other words, the amino acid sequenceof SEQ ID NO:4. In some embodiments, the IL-15Rα protein has the aminoacid sequence of SEQ ID NO:4 and an amino acid insertion selected fromthe group consisting of D96, P97, A98, D96/P97, D96/C97, D96/P97/A98,D96/P97/C98, and D96/C97/A98, wherein the amino acid position isrelative to full-length human IL-15Rα protein or SEQ ID NO:3. Forinstance, amino acid(s) such as D (e.g., Asp), P (e.g., Pro), A (e.g.,Ala), DP (e.g., Asp-Pro), DC (e.g., Asp-Cys), DPA (e.g., Asp-Pro-Ala),DPC (e.g., Asp-Pro-Cys), or DCA (e.g., Asp-Cys-Ala) can be added to theC-terminus of the IL-15Rα protein of SEQ ID NO:4. In some embodiments,the IL-15Rα protein has the amino acid sequence of SEQ ID NO:4 and oneor more amino acid substitutions selected from the group consisting ofK34C, A37C, G38C, S40C, and L42C, wherein the amino acid position isrelative to SEQ ID NO:4. The IL-15Rα protein can have 1, 2, 3, 4, 5, 6,7, 8 or more amino acid mutations (e.g., substitutions, insertionsand/or deletions).

In some embodiments, an IL-15 protein is attached to the N-terminus ofan Fc domain, and an IL-15Rα protein is attached to the N-terminus ofthe IL-15 protein. In other embodiments, an IL-15Rα protein is attachedto the N-terminus of an Fc domain and the IL-15Rα protein isnon-covalently attached to an IL-15 protein. In yet other embodiments,an IL-15Rα protein is attached to the C-terminus of an Fc domain and theIL-15Rα protein is non-covalently attached to an IL-15 protein.

In some embodiments, the IL-15 protein and IL-15Rα protein are attachedtogether via a linker (“scIL-15/Rα”). Optionally, the proteins are notattached via a linker, and utilize either native self-assembly ordisulfide bonds as outlined herein. In other embodiments, the IL-15protein and IL-15Rα protein are noncovalently attached. In someembodiments, the IL-15 protein is attached to an Fc domain via a linker.In other embodiments, the IL-15Rα protein is attached to an Fc domainvia a linker. Optionally, a linker is not used to attach the IL-15protein or IL-15Rα protein to the Fc domain.

In some instances, the PD-1 ABD is covalently attached to the N-terminusof an Fc domain via a linker, such as a domain linker.

In some embodiments, the linker is a “domain linker”, used to link anytwo domains as outlined herein together. While any suitable linker canbe used, many embodiments utilize a glycine-serine polymer, includingfor example (GS)n (SEQ ID NO: 7), (GSGGS)n (SEQ ID NO: 8), (GGGGS)n (SEQID NO: 9), and (GGGS)n (SEQ ID NO: 10), where n is an integer of atleast 1 (and generally from 1 to 2 to 3 to 4 to 5) as well as anypeptide sequence that allows for recombinant attachment of the twodomains with sufficient length and flexibility to allow each domain toretain its biological function. In some cases, and with attention beingpaid to “strandedness”, as outlined below, charged domain linkers can beused as discussed herein and shown in FIG. 4A-4B.

VI. PD-1 Antibody Monomers

Therapeutic antibodies directed against immune checkpoint inhibitorssuch as PD-1 are showing great promise in limited circumstances in theclinic for the treatment of cancer. Cancer can be considered as aninability of the patient to recognize and eliminate cancerous cells. Inmany instances, these transformed (e.g., cancerous) cells counteractimmunosurveillance. There are natural control mechanisms that limitT-cell activation in the body to prevent unrestrained T-cell activity,which can be exploited by cancerous cells to evade or suppress theimmune response. Restoring the capacity of immune effectorcells—especially T cells—to recognize and eliminate cancer is the goalof immunotherapy. The field of immuno-oncology, sometimes referred to as“immunotherapy” is rapidly evolving, with several recent approvals of Tcell checkpoint inhibitory antibodies such as Yervoy, Keytruda andOpdivo. These antibodies are generally referred to as “checkpointinhibitors” because they block normally negative regulators of T cellimmunity. It is generally understood that a variety of immunomodulatorysignals, both costimulatory and coinhibitory, can be used to orchestratean optimal antigen-specific immune response.

Generally, these monoclonal antibodies bind to checkpoint inhibitorproteins such as CTLA-4 and PD-1, which under normal circumstancesprevent or suppress activation of cytotoxic T cells (CTLs). Byinhibiting the checkpoint protein, for example through the use ofantibodies that bind these proteins, an increased T cell responseagainst tumors can be achieved. That is, these cancer checkpointproteins suppress the immune response; when the proteins are blocked,for example using antibodies to the checkpoint protein, the immunesystem is activated, leading to immune stimulation, resulting intreatment of conditions such as cancer and infectious disease.

The present invention relates to the generation of bispecificheterodimeric proteins that bind to a PD-1 and cells expressing IL-2Rβand the common gamma chain (γc; CD132). The bispecific heterodimericprotein can include an antibody monomer of any useful antibody formatthat can bind to an immune checkpoint antigen. In some embodiments, theantibody monomer includes a Fab or a scFv linked to an Fc domain. Insome cases, the PD-1 antibody monomer contains an anti-PD1(VH)-CH1-Fcand an anti-PD-1 VL-Ckappa. In some cases, the PD-1 antibody monomercontains an anti-PD-1 scFv-Fc. Exemplary embodiments of such antibodyfragments are provided in XENP21480 (chain 2; FIG. 64A), XENP22022(chains 2 and 3; FIG. 64D), XENP22112 (chains 1 and 4; FIG. 64E),XENP22641 (chains 1 and 3; FIG. 64F), XENP22642 (chains 1-3; FIG. 64H),and XENP22644 (chains 1-3; FIG. 64I).

The ABD can be in a variety of formats, such as in a Fab format or in anscFv format. Exemplary ABDs for use in the present invention aredisclosed in U.S. 62/353,511, the contents are hereby incorporated inits entirety for all purposes.

Suitable ABDs that bind PD-1 are shown in FIGS. 11 and 12 of U.S.62/353,511, as well as those outlined in FIGS. 13 and 14 herein. As willbe appreciated by those in the art, suitable ABDs can comprise a set of6 CDRs as depicted in these Figures, either as they are underlined or,in the case where a different numbering scheme is used as describedabove, as the CDRs that are identified using other alignments within thevh and vl sequences of FIGS. 11 and 12 of U.S. 62/353,511. Suitable ABDscan also include the entire vh and vl sequences as depicted in theseFigures, used as scFvs or as Fabs. Specific scFv sequences are shown inFIG. 11 U.S. 62/353,511, with a particular charged linker, althoughother linkers, such as those depicted in FIG. 7, can also be used. Inmany of the embodiments herein that contain an Fv to PD-1, it is thescFv monomer that binds PD-1. In U.S. 62/353,511, FIG. 11 showspreferred scFv sequences, and FIG. 12 depicts suitable Fab sequences,although as discussed herein, vh and vl of can be used in eitherconfiguration.

In addition, the antibodies of the invention include those that bind toeither the same epitope as the antigen binding domains outlined herein,or compete for binding with the antigen binding domains outlined herein.In some embodiments, the bispecific checkpoint antibody can contain oneof the ABDs outlined herein and a second ABD that competes for bindingwith one of the ABDs outlined herein. In some embodiments both ABDscompete for binding with the corresponding ABD outlined herein. Bindingcompetition is generally determined using Biacore assays as outlinedherein.

B. Antibodies

As is discussed below, the term “antibody” is used generally. Antibodiesthat find use in the present invention can take on a number of formatsas described herein, including traditional antibodies as well asantibody derivatives, fragments and mimetics, described herein anddepicted in the figures. The present invention provides antibodyfusionproteins containing a checkpoint antigen binding domain and an Fcdomain. In some embodiments, the antibody fusion protein forms abispecific heterodimeric protein with an IL-15/IL-15Rα Fc fusion proteindescribed herein. In other embodiments, the antibody fusion proteinforms a bispecific heterodimeric protein with another antibody fusionprotein comprising a checkpoint antigen binding domain and an Fc domain.Exemplary embodiments of such bispecific heterodimeric proteins include,but are not limited to, XENP21480, XENP22022, XENP22112, XENP22641,XENP22642, and XENP22644.

Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). Human light chains areclassified as kappa and lambda light chains. The present invention isdirected to antibodies or antibody fragments (antibody monomers) thatgenerally are based on the IgG class, which has several subclasses,including, but not limited to IgG1, IgG2, IgG3, and IgG4. In general,IgG1, IgG2 and IgG4 are used more frequently than IgG3. It should benoted that IgG1 has different allotypes with polymorphisms at 356 (D orE) and 358 (L or M). The sequences depicted herein use the 356D/358Mallotype, however the other allotype is included herein. That is, anysequence inclusive of an IgG1 Fc domain included herein can have356E/358L replacing the 356D/358M allotype.

In addition, many of the sequences herein have at least one thecysteines at position 220 replaced by a serine; generally this is the onthe “scFv monomer” side for most of the sequences depicted herein,although it can also be on the “Fab monomer” side, or both, to reducedisulfide formation. Specifically included within the sequences hereinare one or both of these cysteines replaced (C220S).

Thus, “isotype” as used herein is meant any of the subclasses ofimmunoglobulins defined by the chemical and antigenic characteristics oftheir constant regions. It should be understood that therapeuticantibodies can also comprise hybrids of isotypes and/or subclasses. Forexample, as shown in US Publ. Appl. No. 2009/0163699, incorporated byreference, the present invention covers pI engineering of IgG1/G2hybrids.

The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition, generally referred to in the art and herein as the “Fvdomain” or “Fv region”. In the variable region, three loops are gatheredfor each of the V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant“Variable” refers to the fact that certain segments of the variableregion differ extensively in sequence among antibodies. Variabilitywithin the variable region is not evenly distributed. Instead, the Vregions consist of relatively invariant stretches called frameworkregions (FRs) of 15-30 amino acids separated by shorter regions ofextreme variability called “hypervariable regions” that are each 9-15amino acids long or longer.

Each VH and VL is composed of three hypervariable regions(“complementary determining regions,” “CDRs”) and four FRs, arrangedfrom amino-terminus to carboxy-terminus in the following order:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The hypervariable region generally encompasses amino acid residues fromabout amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56(LCDR2) and 89-97 (LCDR3) in the light chain variable region and aroundabout 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102(HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) and/or thoseresidues forming a hypervariable loop (e.g. residues 26-32 (LCDR1),50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chainvariable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.Specific CDRs of the invention are described below.

As will be appreciated by those in the art, the exact numbering andplacement of the CDRs can be different among different numberingsystems. However, it should be understood that the disclosure of avariable heavy and/or variable light sequence includes the disclosure ofthe associated (inherent) CDRs. Accordingly, the disclosure of eachvariable heavy region is a disclosure of the vhCDRs (e.g. vhCDR1, vhCDR2and vhCDR3) and the disclosure of each variable light region is adisclosure of the vlCDRs (e.g. vlCDR1, vlCDR2 and vlCDR3).

A useful comparison of CDR numbering is as below, see Lafranc et al.,Dev. Comp. Immunol. 27(1):55-77 (2003):

TABLE 1 Kabat + Chothia IMGT Kabat AbM Chothia Contact Xencor vhCDR126-35  27-38 31-35 26-35 26-32 30-35  27-35 vhCDR2 50-65  56-65 50-6550-58 52-56 47-58  54-61 vhCDR3 95-102 105-117 95-102 95-102 95-10293-101 103-116 vlCDR1 24-34  27-38 24-34 24-34 24-34 30-36  27-38 vlCDR250-56  56-65 50-56 50-56 50-56 46-55  56-62 vlCDR3 89-97 105-117 89-9789-97 89-97 89-96  97-105

Throughout the present specification, the Kabat numbering system isgenerally used when referring to a residue in the variable domain(approximately, residues 1-107 of the light chain variable region andresidues 1-113 of the heavy chain variable region) and the EU numberingsystem for Fc regions (e.g., Kabat et al., supra (1991)).

The present invention provides a large number of different CDR sets. Inthis case, a “full CDR set” comprises the three variable light and threevariable heavy CDRs, e.g. a vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2 andvhCDR3. These can be part of a larger variable light or variable heavydomain, respectfully. In addition, as more fully outlined herein, thevariable heavy and variable light domains can be on separate polypeptidechains, when a heavy and light chain is used (for example when Fabs areused), or on a single polypeptide chain in the case of scFv sequences.

The CDRs contribute to the formation of the antigen-binding, or morespecifically, epitope binding site of antibodies. “Epitope” refers to adeterminant that interacts with a specific antigen binding site in thevariable region of an antibody molecule known as a paratope. Epitopesare groupings of molecules such as amino acids or sugar side chains andusually have specific structural characteristics, as well as specificcharge characteristics. A single antigen may have more than one epitope.

The epitope may comprise amino acid residues directly involved in thebinding (also called immunodominant component of the epitope) and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecifically antigen binding peptide; in other words, the amino acidresidue is within the footprint of the specifically antigen bindingpeptide.

Epitopes may be either conformational or linear. A conformationalepitope is produced by spatially juxtaposed amino acids from differentsegments of the linear polypeptide chain. A linear epitope is oneproduced by adjacent amino acid residues in a polypeptide chain.Conformational and nonconformational epitopes may be distinguished inthat the binding to the former but not the latter is lost in thepresence of denaturing solvents.

An epitope typically includes at least 3, and more usually, at least 5or 8-10 amino acids in a unique spatial conformation. Antibodies thatrecognize the same epitope can be verified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen, for example “binning.” As outlined below,the invention not only includes the enumerated antigen binding domainsand antibodies herein, but those that compete for binding with theepitopes bound by the enumerated antigen binding domains.

The carboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. Kabat et al. collectednumerous primary sequences of the variable regions of heavy chains andlight chains. Based on the degree of conservation of the sequences, theyclassified individual primary sequences into the CDR and the frameworkand made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5thedition, NIH publication, No. 91-3242, E. A. Kabat et al., entirelyincorporated by reference).

In the IgG subclass of immunoglobulins, there are several immunoglobulindomains in the heavy chain. By “immunoglobulin (Ig) domain” herein ismeant a region of an immunoglobulin having a distinct tertiarystructure. Of interest in the present invention are the heavy chaindomains, including, the constant heavy (CH) domains and the hingedomains. In the context of IgG antibodies, the IgG isotypes each havethree CH regions. Accordingly, “CH” domains in the context of IgG are asfollows: “CH1” refers to positions 118-220 according to the EU index asin Kabat. “CH2” refers to positions 237-340 according to the EU index asin Kabat, and “CH3” refers to positions 341-447 according to the EUindex as in Kabat. As shown herein and described below, the pI variantscan be in one or more of the CH regions, as well as the hinge region,discussed below.

Another type of Ig domain of the heavy chain is the hinge region. By“hinge” or “hinge region” or “antibody hinge region” or “immunoglobulinhinge region” herein is meant the flexible polypeptide comprising theamino acids between the first and second constant domains of anantibody. Structurally, the IgG CH1 domain ends at EU position 220, andthe IgG CH2 domain begins at residue EU position 237. Thus for IgG theantibody hinge is herein defined to include positions 221 (D221 in IgG1)to 236 (G236 in IgG1), wherein the numbering is according to the EUindex as in Kabat. In some embodiments, for example in the context of anFc region, the lower hinge is included, with the “lower hinge” generallyreferring to positions 226 or 230. As noted herein, pI variants can bemade in the hinge region as well.

The light chain generally comprises two domains, the variable lightdomain (containing the light chain CDRs and together with the variableheavy domains forming the Fv region), and a constant light chain region(often referred to as CL or CK).

Another region of interest for additional substitutions, outlined above,is the Fc region.

Thus, the present invention provides different antibody domains. Asdescribed herein and known in the art, the heterodimeric antibodies ofthe invention comprise different domains within the heavy and lightchains, which can be overlapping as well. These domains include, but arenot limited to, the Fc domain, the CH1 domain, the CH2 domain, the CH3domain, the hinge domain, the heavy constant domain (CH1-hinge-Fc domainor CH1-hinge-CH2-CH3), the variable heavy domain, the variable lightdomain, the light constant domain, Fab domains and scFv domains.

Thus, the “Fc domain” includes the —CH2-CH3 domain, and optionally ahinge domain. In the embodiments herein, when a scFv is attached to anFc domain, it is the C-terminus of the scFv construct that is attachedto all or part of the hinge of the Fc domain; for example, it isgenerally attached to the sequence EPKS (SEQ ID NO: 15) which is thebeginning of the hinge. The heavy chain comprises a variable heavydomain and a constant domain, which includes a CH1-optional hinge-Fcdomain comprising a CH2-CH3. The light chain comprises a variable lightchain and the light constant domain. A scFv comprises a variable heavychain, an scFv linker, and a variable light domain. In most of theconstructs and sequences outlined herein, C-terminus of the variablelight chain is attached to the N-terminus of the scFv linker, theC-terminus of which is attached to the N-terminus of a variable heavychain (N-vh-linker-vl-C) although that can be switched(N-vl-linker-vh-C).

Some embodiments of the invention comprise at least one scFv domain,which, while not naturally occurring, generally includes a variableheavy domain and a variable light domain, linked together by a scFvlinker. As outlined herein, while the scFv domain is generally from N-to C-terminus oriented as vh-scFv linker-vl, this can be reversed forany of the scFv domains (or those constructed using vh and vl sequencesfrom Fabs), to vl-scFv linker-vh, with optional linkers at one or bothends depending on the format (see generally FIGS. 4A-4B of U.S.62/353,511).

As shown herein, there are a number of suitable scFv linkers that can beused, including traditional peptide bonds, generated by recombinanttechniques. The linker peptide may predominantly include the followingamino acid residues: Gly, Ser, Ala, or Thr. The linker peptide shouldhave a length that is adequate to link two molecules in such a way thatthey assume the correct conformation relative to one another so thatthey retain the desired activity. In one embodiment, the linker is fromabout 1 to 50 amino acids in length, preferably about 1 to 30 aminoacids in length. In one embodiment, linkers of 1 to 20 amino acids inlength may be used, with from about 5 to about 10 amino acids findinguse in some embodiments. Useful linkers include glycine-serine polymers,including for example (GS)n (SEQ ID NO: 11), (GSGGS)n (SEQ ID NO: 12),(GGGGS)n (SEQ ID NO: 13), and (GGGS)n (SEQ ID NO: 14), where n is aninteger of at least one (and generally from 3 to 4), glycine-alaninepolymers, alanine-serine polymers, and other flexible linkers.Alternatively, a variety of nonproteinaceous polymers, including but notlimited to polyethylene glycol (PEG), polypropylene glycol,polyoxyalkylenes, or copolymers of polyethylene glycol and polypropyleneglycol, may find use as linkers, that is may find use as linkers.

Other linker sequences may include any sequence of any length of CL/CH1domain but not all residues of CL/CH1 domain; for example the first 5-12amino acid residues of the CL/CH1 domains. Linkers can be derived fromimmunoglobulin light chain, for example Cκ or Cλ. Linkers can be derivedfrom immunoglobulin heavy chains of any isotype, including for exampleCγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Linker sequences may alsobe derived from other proteins such as Ig-like proteins (e.g. TCR, FcR,KIR), hinge region-derived sequences, and other natural sequences fromother proteins.

In some embodiments, the linker is a “domain linker”, used to link anytwo domains as outlined herein together. While any suitable linker canbe used, many embodiments utilize a glycine-serine polymer, includingfor example (GS)n (SEQ ID NO: 7), (GSGGS)n (SEQ ID NO: 8), (GGGGS)n (SEQID NO: 9), and (GGGS)n (SEQ ID NO: 10), where n is an integer of atleast one (and generally from 3 to 4 to 5) as well as any peptidesequence that allows for recombinant attachment of the two domains withsufficient length and flexibility to allow each domain to retain itsbiological function. In some cases, and with attention being paid to“strandedness”, as outlined below, charged domain linkers, as used insome embodiments of scFv linkers can be used.

In some embodiments, the scFv linker is a charged scFv linker, a numberof which are shown in FIG. 4A of U.S. 62/353,511. Accordingly, thepresent invention further provides charged scFv linkers, to facilitatethe separation in pI between a first and a second monomer (e.g., anIL-15/IL-15Rα monomer and PD-1 ABD monomer). That is, by incorporating acharged scFv linker, either positive or negative (or both, in the caseof scaffolds that use scFvs on different monomers), this allows themonomer comprising the charged linker to alter the pI without makingfurther changes in the Fc domains. These charged linkers can besubstituted into any scFv containing standard linkers. Again, as will beappreciated by those in the art, charged scFv linkers are used on thecorrect “strand” or monomer, according to the desired changes in pI. Forexample, as discussed herein, to make triple F format heterodimericantibody, the original pI of the Fv region for each of the desiredantigen binding domains are calculated, and one is chosen to make anscFv, and depending on the pI, either positive or negative linkers arechosen.

Charged domain linkers can also be used to increase the pI separation ofthe monomers of the invention as well, and thus those included in FIG.4A can be used in any embodiment herein where a linker is utilized.

In one embodiment, the antibody is an antibody fragment, as long as itcontains at least one constant domain which can be engineered to produceheterodimers, such as pI engineering. Other antibody fragments that canbe used include fragments that contain one or more of the CH1, CH2, CH3,hinge and CL domains of the invention that have been pI engineered. Inparticular, the formats depicted in FIGS. 8A-8C and 16A-16C areantibodies, referred to as “heterodimeric antibodies” or “bispecificheterodimer fusion proteins”, meaning that the protein has at least twoassociated Fc sequences self-assembled into a heterodimeric Fc domainand at least one Fv regions, whether as Fabs or as scFvs.

C. Chimeric and Humanized Antibodies

In some embodiments, the antibodies herein can be derived from a mixturefrom different species, e.g., a chimeric antibody and/or a humanizedantibody. In general, both “chimeric antibodies” and “humanizedantibodies” refer to antibodies that combine regions from more than onespecies. For example, “chimeric antibodies” traditionally comprisevariable region(s) from a mouse (or rat, in some cases) and the constantregion(s) from a human. “Humanized antibodies” generally refer tonon-human antibodies that have had the variable-domain framework regionsswapped for sequences found in human antibodies. Generally, in ahumanized antibody, the entire antibody, except the CDRs, is encoded bya polynucleotide of human origin or is identical to such an antibodyexcept within its CDRs. The CDRs, some or all of which are encoded bynucleic acids originating in a non-human organism, are grafted into thebeta-sheet framework of a human antibody variable region to create anantibody, the specificity of which is determined by the engrafted CDRs.The creation of such antibodies is described in, e.g., WO 92/11018,Jones, 1986, Nature 321:522-525, Verhoeyen et al., 1988, Science239:1534-1536, all entirely incorporated by reference. “Backmutation” ofselected acceptor framework residues to the corresponding donor residuesis often required to regain affinity that is lost in the initial graftedconstruct (U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762;6,180,370; 5,859,205; 5,821,337; 6,054,297; 6,407,213, all entirelyincorporated by reference). The humanized antibody optimally also willcomprise at least a portion of an immunoglobulin constant region,typically that of a human immunoglobulin, and thus will typicallycomprise a human Fc region Humanized antibodies can also be generatedusing mice with a genetically engineered immune system. Roque et al.,2004, Biotechnol. Prog. 20:639-654, entirely incorporated by reference.A variety of techniques and methods for humanizing and reshapingnon-human antibodies are well known in the art (See Tsurushita &Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biologyof B Cells, 533-545, Elsevier Science (USA), and references citedtherein, all entirely incorporated by reference). Humanization methodsinclude but are not limited to methods described in Jones et al., 1986,Nature 321:522-525; Riechmann et al., 1988; Nature 332:323-329;Verhoeyen et al., 1988, Science, 239:1534-1536; Queen et al., 1989, ProcNatl Acad Sci, USA 86:10029-33; He et al., 1998, J. Immunol. 160:1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA 89:4285-9, Prestaet al., 1997, Cancer Res. 57(20):4593-9; Gorman et al., 1991, Proc.Natl. Acad. Sci. USA 88:4181-4185; O'Connor et al., 1998, Protein Eng11:321-8, all entirely incorporated by reference. Humanization or othermethods of reducing the immunogenicity of nonhuman antibody variableregions may include resurfacing methods, as described for example inRoguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973, entirelyincorporated by reference. In certain embodiments, the antibodies of theinvention comprise a heavy chain variable region from a particulargermline heavy chain immunoglobulin gene and/or a light chain variableregion from a particular germline light chain immunoglobulin gene. Forexample, such antibodies may comprise or consist of a human antibodycomprising heavy or light chain variable regions that are “the productof” or “derived from” a particular germline sequence. A human antibodythat is “the product of” or “derived from” a human germlineimmunoglobulin sequence can be identified as such by comparing the aminoacid sequence of the human antibody to the amino acid sequences of humangermline immunoglobulins and selecting the human germline immunoglobulinsequence that is closest in sequence (i.e., greatest % identity) to thesequence of the human antibody. A human antibody that is “the productof” or “derived from” a particular human germline immunoglobulinsequence may contain amino acid differences as compared to the germlinesequence, due to, for example, naturally-occurring somatic mutations orintentional introduction of site-directed mutation. However, a humanizedantibody typically is at least 90% identical in amino acids sequence toan amino acid sequence encoded by a human germline immunoglobulin geneand contains amino acid residues that identify the antibody as beingderived from human sequences when compared to the germlineimmunoglobulin amino acid sequences of other species (e.g., murinegermline sequences). In certain cases, a humanized antibody may be atleast 95, 96, 97, 98 or 99%, or even at least 96%, 97%, 98%, or 99%identical in amino acid sequence to the amino acid sequence encoded bythe germline immunoglobulin gene. Typically, a humanized antibodyderived from a particular human germline sequence will display no morethan 10-20 amino acid differences from the amino acid sequence encodedby the human germline immunoglobulin gene (prior to the introduction ofany skew, pI and ablation variants herein; that is, the number ofvariants is generally low, prior to the introduction of the variants ofthe invention). In certain cases, the humanized antibody may display nomore than 5, or even no more than 4, 3, 2, or 1 amino acid differencefrom the amino acid sequence encoded by the germline immunoglobulin gene(again, prior to the introduction of any skew, pI and ablation variantsherein; that is, the number of variants is generally low, prior to theintroduction of the variants of the invention). In one embodiment, theparent antibody has been affinity matured, as is known in the art.Structure-based methods may be employed for humanization and affinitymaturation, for example as described in U.S. Ser. No. 11/004,590.Selection based methods may be employed to humanize and/or affinitymature antibody variable regions, including but not limited to methodsdescribed in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al.,1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol.Chem. 271(37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci.USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering16(10):753-759, all entirely incorporated by reference. Otherhumanization methods may involve the grafting of only parts of the CDRs,including but not limited to methods described in U.S. Ser. No.09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis etal., 2002, J. Immunol. 169:3076-3084, all entirely incorporated byreference.

VII. Useful Embodiments of the Invention

The present invention provides a bispecific heterodimeric proteincomprising a fusion protein and an antibody fusion protein. The fusionprotein comprises a first protein domain, a second protein domain, and afirst Fc domain. In some cases, the first protein domain is covalentlyattached to the N-terminus of the second protein domain using a firstdomain linker, the second protein domain is covalently attached to theN-terminus of the first Fc domain using a second domain linker, and thefirst protein domain comprises an IL-15Rα protein and the second proteindomain comprises an IL-15 protein. The antibody fusion protein comprisesa PD-1 antigen binding domain and a second Fc domain such that the PD-1antigen binding domain is covalently attached to the N-terminus of thesecond Fc domain, and the PD-1 antigen binding domain is a single chainvariable fragment (scFv) or a Fab fragment. In some embodiments, thefirst and the second Fc domains have a set of amino acid substitutionsselected from the group consisting ofS267K/L368D/K370S:S267K/LS364K/E357Q; S364K/E357Q:L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411T/E360E/Q362E:D401K;L368D/K370S:S364K/E357L and K370S:S364K/E357Q, according to EUnumbering. In some instances, the first and/or the second Fc domainshave an additional set of amino acid substitutions comprisingQ295E/N384D/Q418E/N421D, according to EU numbering. In some cases, thefirst and/or the second Fc domains have an additional set of amino acidsubstitutions consisting of G236R/L328R,E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K,E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G and E233P/L234V/L235A/G236del,according to EU numbering.

In some embodiments, the IL-15 protein has a polypeptide sequenceselected from the group consisting of SEQ ID NO: 1 (full-length humanIL-15) and SEQ ID NO:2 (truncated human IL-15), and the IL-15Rα proteinhas a polypeptide sequence selected from the group consisting of SEQ IDNO:3 (full-length human IL-15Rα) and SEQ ID NO:4 (sushi domain of humanIL-15Rα). The IL-15 protein and the IL-15Rα protein can have a set ofamino acid substitutions selected from the group consisting ofE87C:D96/P97/C98; E87C:D96/C97/A98; V49C:S40C; L52C:S40C; E89C:K34C;Q48C:G38C; E53C:L42C; C42S:A37C; and L45C:A37C, respectively.

In some embodiments, said PD-1 antigen binding domain comprises ananti-PD-1 scFv or an anti-PD-1 Fab.

In some embodiments, the first fusion protein has a polypeptide sequenceof SEQ ID NO: 233 (16478) and said Fab of the PD-1 antigen bindingdomain has polypeptide sequences of SEQ ID NO:XX (14833) and SEQ IDNO:XX (14812). In other embodiments, the bispecific heterodimericprotein can be XENP21480, XENP22022, or those depicted in FIGS. 8A, 8B,13, and 14 .

Also provided are nucleic acid compositions encoding the fusion proteinor the antibody fusion protein described herein. In some instances, anexpression vector comprising one or more nucleic acid compositionsdescribed herein. In some embodiments, a host cell comprising one or twoexpression vectors outlined herein is provided.

The present invention also provides a bispecific heterodimeric proteincomprising a fusion protein, a second protein that is noncovalentlyattached to the first protein domain of the fusion protein, an antibodyfusion protein. In some embodiments, the fusion protein comprises afirst protein domain and a first Fc domain. The first protein domain canbe covalently attached to the N-terminus of the first Fc domain using adomain linker and the first protein domain can include an IL-15Rαprotein such as that of SEQ ID NO:3 or 4. In some instances, the secondprotein domain which is noncovalently attached to said first proteindomain includes an IL-15 protein (SEQ ID NO: 1) or a fragment of theIL-15 protein (SEQ ID NO:2). The IL-15 protein and the IL-15Rα proteincan have a set of amino acid substitutions selected from the groupconsisting of E87C:D96/P97/C98; E87C: D96/C97/A98; V49C:S40C; L52C:S40C;E89C:K34C; Q48C:G38C; E53C:L42C; C42S:A37C; and L45C:A37C, respectively.

In some embodiments, the antibody fusion protein comprises an PD-1antigen binding domain and a second Fc domain. In some instances, thePD-1 antigen binding domain is covalently attached to the N-terminus ofthe second Fc domain. The PD-1 antigen binding domain can be singlechain variable fragment (scFv) or a Fab fragment. In some cases, thePD-1 antigen binding domain comprises an anti-PD-1 scFv or an anti-PD-1Fab.

In some embodiments, the first and the second Fc domains of thebispecific heterodimeric proteins have a set of amino acid substitutionsselected from the group consisting ofS267K/L368D/K370S:S267K/LS364K/E357Q; S364K/E357Q:L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411T/E360E/Q362E:D401K;L368D/K370S:S364K/E357L and K370S:S364K/E357Q, according to EUnumbering. In some instances, the first and/or the second Fc domainshave an additional set of amino acid substitutions comprisingQ295E/N384D/Q418E/N421D, according to EU numbering. In some cases, thefirst and/or the second Fc domains have an additional set of amino acidsubstitutions consisting of G236R/L328R,E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K,E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G and E233P/L234V/L235A/G236del,according to EU numbering.

In certain embodiments, the bispecific heterodimeric protein comprisesthe fusion protein having a polypeptide sequence of SEQ ID NOS 230, 240,and 421 (16481), the second protein domain having a polypeptide sequenceof SEQ ID NOS 238, 243, 256, and 265 (16484), and the Fab of the PD-1antigen binding domain having polypeptide sequences of SEQ ID NO:XX(14833) and SEQ ID NO:XX (14812). In some embodiments, the bispecificheterodimer protein comprises the fusion protein having a polypeptidesequence of SEQ ID NO:XX (17584), the second protein domain having apolypeptide sequence of SEQ ID NOS 314, 319, 336, 340, and 348 (17074),and the Fab of the PD-1 antigen binding domain having polypeptidesequences of SEQ ID NO:XX (14833) and SEQ ID NO:XX (14812). In someinstances, the bispecific heterodimer protein is selected from the groupconsisting of XENP22112, XENP22641, and those depicted in FIGS. 64A-64K.

Also provided are nucleic acid compositions encoding the fusion protein,the second protein domain, and/or the antibody fusion protein describedherein. In some instances, an expression vector comprising one or two orthree nucleic acid compositions described herein. In some embodiments, ahost cell comprising one or two or three expression vectors outlinedherein is provided.

The present invention also provides a bispecific heterodimeric proteincomprising a first antibody fusion protein, a second antibody fusionprotein, and a second protein domain that is noncovalently attached tothe first protein domain of the second antibody fusion protein.

In some embodiments, the first antibody fusion protein comprises a firstPD-1 antigen binding domain and a first Fc domain such that the firstPD-1 antigen binding domain is covalently attached to the N-terminus ofthe first Fc domain via a first domain linker. The first PD-1 antigenbinding domain can be a single chain variable fragment (scFv) or a Fabfragment.

In some embodiments, the second antibody fusion protein comprises asecond PD-1 antigen binding domain, a second Fc domain, and a firstprotein domain such that the second PD-1 antigen binding domain iscovalently attached to the N-terminus of said second Fc domain via asecond domain linker. The first protein domain can be covalentlyattached to the C-terminus of the second Fc domain via a third domainlinker. In some instance, the second PD-1 antigen binding domain is asingle chain variable fragment (scFv) or a Fab fragment. The firstprotein domain can include an IL-15Rα protein, such as the protein setforth in SEQ ID NOS: 3 or 4.

In some embodiments, the second protein domain is noncovalently attachedto the first protein domain of the second antibody fusion protein andsuch second protein domain comprises an IL-15 protein, e.g., the proteinset forth in SEQ ID NOS: 1 or 2.

The IL-15 protein and the IL-15Rα protein can have a set of amino acidsubstitutions selected from the group consisting of E87C:D96/P97/C98;E87C:D96/C97/A98; V49C:S40C; L52C:S40C; E89C:K34C; Q48C:G38C; E53C:L42C;C42S:A37C; and L45C:A37C, respectively.

In some embodiments, the first and the second Fc domains of thebispecific heterodimeric proteins have a set of amino acid substitutionsselected from the group consisting ofS267K/L368D/K370S:S267K/LS364K/E357Q; S364K/E357Q:L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411T/E360E/Q362E:D401K;L368D/K370S:S364K/E357L and K370S:S364K/E357Q, according to EUnumbering. In some instances, the first and/or the second Fc domainshave an additional set of amino acid substitutions comprisingQ295E/N384D/Q418E/N421D, according to EU numbering. In some cases, thefirst and/or the second Fc domains have an additional set of amino acidsubstitutions consisting of G236R/L328R,E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K,E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G and E233P/L234V/L235A/G236del,according to EU numbering.

The first PD-1 antigen binding domain of the first antibody fusionprotein can include an anti-PD-1 scFv or an anti-PD-1 Fab. The secondPD-1 antigen binding domain of the second antibody fusion protein caninclude an anti-PD-1 scFv or an anti-PD-1 Fab.

In some instances, the bispecific heterodimeric protein of the presentinvention contains a first antibody fusion protein having polypeptidesequences of SEQ ID NO:XX (17599) and SEQ ID NO: XX (9016), a secondantibody fusion protein having polypeptide sequences of SEQ ID NO: 233(16478) and SEQ ID NO: XX (9016), and a second protein domain having apolypeptide sequence of SEQ ID NOS 238, 243, 256, and 265 (16484). Inother instances, the bispecific heterodimeric protein of the presentinvention contains a first antibody fusion protein having polypeptidesequences of SEQ ID NO:XX (17601) and SEQ ID NO: XX (9016), a secondantibody fusion protein having polypeptide sequences of SEQ ID NO:XX(9018) and SEQ ID NO: XX (9016), and a second protein domain having apolypeptide sequence of SEQ ID NOS 314, 319, 336, 340, and 348 (17074).The bispecific heterodimeric protein can be XENP22642 or XENP22644 orthose depicts in FIGS. 16B, 16C, 18 and 19 .

VIII. Nucleic Acids of the Invention

The invention further provides nucleic acid compositions encoding thebispecific heterodimeric fusion protein of the invention (or, in thecase of a monomer Fc domain protein, nucleic acids encoding those aswell).

As will be appreciated by those in the art, the nucleic acidcompositions will depend on the format of the bispecific heterodimericfusion protein. Thus, for example, when the format requires three aminoacid sequences, three nucleic acid sequences can be incorporated intoone or more expression vectors for expression. Similarly, some formatsonly two nucleic acids are needed; again, they can be put into one ortwo expression vectors.

As is known in the art, the nucleic acids encoding the components of theinvention can be incorporated into expression vectors as is known in theart, and depending on the host cells used to produce the bispecificheterodimeric fusion proteins of the invention. Generally the nucleicacids are operably linked to any number of regulatory elements(promoters, origin of replication, selectable markers, ribosomal bindingsites, inducers, etc.). The expression vectors can be extra-chromosomalor integrating vectors.

The nucleic acids and/or expression vectors of the invention are thentransformed into any number of different types of host cells as is wellknown in the art, including mammalian, bacterial, yeast, insect and/orfungal cells, with mammalian cells (e.g. CHO cells), finding use in manyembodiments.

In some embodiments, nucleic acids encoding each monomer, as applicabledepending on the format, are each contained within a single expressionvector, generally under different or the same promoter controls. Inembodiments of particular use in the present invention, each of thesetwo or three nucleic acids are contained on a different expressionvector.

The bispecific heterodimeric fusion protein of the invention are made byculturing host cells comprising the expression vector(s) as is wellknown in the art. Once produced, traditional fusion protein or antibodypurification steps are done, including an ion exchange chromotographystep. As discussed herein, having the pIs of the two monomers differ byat least 0.5 can allow separation by ion exchange chromatography orisoelectric focusing, or other methods sensitive to isoelectric point.That is, the inclusion of pI substitutions that alter the isoelectricpoint (pI) of each monomer so that such that each monomer has adifferent pI and the heterodimer also has a distinct pI, thusfacilitating isoelectric purification of the heterodimer (e.g., anionicexchange columns, cationic exchange columns). These substitutions alsoaid in the determination and monitoring of any contaminating homodimerspost-purification (e.g., IEF gels, cIEF, and analytical IEX columns).

IX. Biological and Biochemical Functionality of Bispecific ImmuneCheckpoint Antibody×IL-15/IL-15Rα Heterodimeric Immunomodulatory FusionProteins

Generally the bispecific heterodimeric fusion proteins of the inventionare administered to patients with cancer, and efficacy is assessed, in anumber of ways as described herein. Thus, while standard assays ofefficacy can be run, such as cancer load, size of tumor, evaluation ofpresence or extent of metastasis, etc., immuno-oncology treatments canbe assessed on the basis of immune status evaluations as well. This canbe done in a number of ways, including both in vitro and in vivo assays.For example, evaluation of changes in immune status (e.g., presence ofICOS+ CD4+ T cells following ipi treatment) along with “old fashioned”measurements such as tumor burden, size, invasiveness, LN involvement,metastasis, etc. can be done. Thus, any or all of the following can beevaluated: the inhibitory effects of PVRIG on CD4⁺ T cell activation orproliferation, CD8⁺ T (CTL) cell activation or proliferation, CD8⁺ Tcell-mediated cytotoxic activity and/or CTL mediated cell depletion, NKcell activity and NK mediated cell depletion, the potentiating effectsof PVRIG on Treg cell differentiation and proliferation and Treg- ormyeloid derived suppressor cell (MDSC)-mediated immunosuppression orimmune tolerance, and/or the effects of PVRIG on proinflammatorycytokine production by immune cells, e.g., IL-2, IFN-γ or TNF-αproduction by T or other immune cells.

In some embodiments, assessment of treatment is done by evaluatingimmune cell proliferation, using for example, CFSE dilution method, Ki67intracellular staining of immune effector cells, and ³H-thymidineincorporation method,

In some embodiments, assessment of treatment is done by evaluating theincrease in gene expression or increased protein levels ofactivation-associated markers, including one or more of: CD25, CD69,CD137, ICOS, PD1, GITR, OX40, and cell degranulation measured by surfaceexpression of CD107A.

In general, gene expression assays are done as is known in the art.

In general, protein expression measurements are also similarly done asis known in the art.

In some embodiments, assessment of treatment is done by assessingcytotoxic activity measured by target cell viability detection viaestimating numerous cell parameters such as enzyme activity (includingprotease activity), cell membrane permeability, cell adherence, ATPproduction, co-enzyme production, and nucleotide uptake activity.Specific examples of these assays include, but are not limited to,Trypan Blue or PI staining, ⁵¹Cr or ³⁵S release method, LDH activity,MTT and/or WST assays, Calcein-AM assay, Luminescent based assay, andothers.

In some embodiments, assessment of treatment is done by assessing T cellactivity measured by cytokine production, measure either intracellularlyin culture supernatant using cytokines including, but not limited to,IFNγ, TNFα, GM-CSF, IL2, IL6, IL4, IL5, IL10, IL3 using well knowntechniques.

Accordingly, assessment of treatment can be done using assays thatevaluate one or more of the following: (i) increases in immune response,(ii) increases in activation of αβ and/or γδ T cells, (iii) increases incytotoxic T cell activity, (iv) increases in NK and/or NKT cellactivity, (v) alleviation of αβ and/or γδ T-cell suppression, (vi)increases in pro-inflammatory cytokine secretion, (vii) increases inIL-2 secretion; (viii) increases in interferon-γ production, (ix)increases in Th1 response, (x) decreases in Th2 response, (xi) decreasesor eliminates cell number and/or activity of at least one of regulatoryT cells (Tregs).

A. Assays to Measure Efficacy

In some embodiments, T cell activation is assessed using a MixedLymphocyte Reaction (MLR) assay as is known in the art. An increase inactivity indicates immunostimulatory activity. Appropriate increases inactivity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in immune response as measured for an example byphosphorylation or de-phosphorylation of different factors, or bymeasuring other post translational modifications. An increase inactivity indicates immunostimulatory activity. Appropriate increases inactivity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in activation of al and/or γδ T cells as measured for anexample by cytokine secretion or by proliferation or by changes inexpression of activation markers like for an example CD137, CD107a, PD1,etc. An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in cytotoxic T cell activity as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in NK and/or NKT cell activity as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by changes in expression of activation markerslike for an example CD107a, etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in αβ and/or γδ T-cell suppression, as measured for an exampleby cytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in pro-inflammatory cytokine secretion as measured for exampleby ELISA or by Luminex or by Multiplex bead based methods or byintracellular staining and FACS analysis or by Alispot etc. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in IL-2 secretion as measured for example by ELISA or byLuminex or by Multiplex bead based methods or by intracellular stainingand FACS analysis or by Alispot etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in interferon-γ production as measured for example by ELISA orby Luminex or by Multiplex bead based methods or by intracellularstaining and FACS analysis or by Alispot etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in Th1 response as measured for an example by cytokinesecretion or by changes in expression of activation markers. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in Th2 response as measured for an example by cytokinesecretion or by changes in expression of activation markers. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases cell number and/or activity of at least one of regulatory Tcells (Tregs), as measured for example by flow cytometry or by IHC. Adecrease in response indicates immunostimulatory activity. Appropriatedecreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in M2 macrophages cell numbers, as measured for example byflow cytometry or by IHC. A decrease in response indicatesimmunostimulatory activity. Appropriate decreases are the same as forincreases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in M2 macrophage pro-tumorigenic activity, as measured for anexample by cytokine secretion or by changes in expression of activationmarkers. A decrease in response indicates immunostimulatory activity.Appropriate decreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in N2 neutrophils increase, as measured for example by flowcytometry or by IHC. A decrease in response indicates immunostimulatoryactivity. Appropriate decreases are the same as for increases, outlinedbelow.

In one embodiment, the signaling pathway assay measures increases ordecreases in N2 neutrophils pro-tumorigenic activity, as measured for anexample by cytokine secretion or by changes in expression of activationmarkers. A decrease in response indicates immunostimulatory activity.Appropriate decreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in inhibition of T cell activation, as measured for an exampleby cytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in inhibition of CTL activation as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in αβ and/or γδT cell exhaustion as measured for an example bychanges in expression of activation markers. A decrease in responseindicates immunostimulatory activity. Appropriate decreases are the sameas for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases αβ and/or γδ T cell response as measured for an example bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in stimulation of antigen-specific memory responses asmeasured for an example by cytokine secretion or by proliferation or bychanges in expression of activation markers like for an example CD45RA,CCR7 etc. An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in apoptosis or lysis of cancer cells as measured for anexample by cytotoxicity assays such as for an example MTT, Cr release,Calcine AM, or by flow cytometry based assays like for an example CFSEdilution or propidium iodide staining etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in stimulation of cytotoxic or cytostatic effect on cancercells. as measured for an example by cytotoxicity assays such as for anexample MTT, Cr release, Calcine AM, or by flow cytometry based assayslike for an example CFSE dilution or propidium iodide staining etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases direct killing of cancer cells as measured for an example bycytotoxicity assays such as for an example MTT, Cr release, Calcine AM,or by flow cytometry based assays like for an example CFSE dilution orpropidium iodide staining etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases Th17 activity as measured for an example by cytokine secretionor by proliferation or by changes in expression of activation markers.An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in induction of complement dependent cytotoxicity and/orantibody dependent cell-mediated cytotoxicity, as measured for anexample by cytotoxicity assays such as for an example MTT, Cr release,Calcine AM, or by flow cytometry based assays like for an example CFSEdilution or propidium iodide staining etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, T cell activation is measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. ForT-cells, increases in proliferation, cell surface markers of activation(e.g. CD25, CD69, CD137, PD1), cytotoxicity (ability to kill targetcells), and cytokine production (e.g. IL-2, IL-4, IL-6, IFNγ, TNF-a,IL-10, IL-17A) would be indicative of immune modulation that would beconsistent with enhanced killing of cancer cells.

In one embodiment, NK cell activation is measured for example by directkilling of target cells like for an example cancer cells or by cytokinesecretion or by changes in expression of activation markers like for anexample CD107a, etc. For NK cells, increases in proliferation,cytotoxicity (ability to kill target cells and increases CD107a,granzyme, and perforin expression), cytokine production (e.g. IFNγ andTNF), and cell surface receptor expression (e.g. CD25) would beindicative of immune modulation that would be consistent with enhancedkilling of cancer cells.

In one embodiment, γδ T cell activation is measured for example bycytokine secretion or by proliferation or by changes in expression ofactivation markers.

In one embodiment, Th1 cell activation is measured for example bycytokine secretion or by changes in expression of activation markers.

Appropriate increases in activity or response (or decreases, asappropriate as outlined above), are increases of 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95% or 98 to 99% percent over the signal ineither a reference sample or in control samples, for example testsamples that do not contain an anti-PVRIG antibody of the invention.Similarly, increases of at least one-, two-, three-, four- or five-foldas compared to reference or control samples show efficacy.

X. Treatments

Once made, the compositions of the invention find use in a number ofoncology applications, by treating cancer, generally by promoting T cellactivation (e.g., T cells are no longer suppressed) with the binding ofthe heterodimeric Fc fusion proteins of the invention.

Accordingly, the bispecific heterodimeric compositions of the inventionfind use in the treatment of these cancers.

A. Bispecific Heterodimeric Protein Compositions for In VivoAdministration

Formulations of the antibodies used in accordance with the presentinvention are prepared for storage by mixing an antibody having thedesired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (as generally outlined inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, buffers, excipients, or stabilizers are nontoxic to recipientsat the dosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

B. Administrative Modalities

The bispecific heterodimeric proteins and chemotherapeutic agents of theinvention are administered to a subject, in accord with known methods,such as intravenous administration as a bolus or by continuous infusionover a period of time.

C. Treatment Modalities

In the methods of the invention, therapy is used to provide a positivetherapeutic response with respect to a disease or condition. By“positive therapeutic response” is intended an improvement in thedisease or condition, and/or an improvement in the symptoms associatedwith the disease or condition. For example, a positive therapeuticresponse would refer to one or more of the following improvements in thedisease: (1) a reduction in the number of neoplastic cells; (2) anincrease in neoplastic cell death; (3) inhibition of neoplastic cellsurvival; (5) inhibition (i.e., slowing to some extent, preferablyhalting) of tumor growth; (6) an increased patient survival rate; and(7) some relief from one or more symptoms associated with the disease orcondition.

Positive therapeutic responses in any given disease or condition can bedetermined by standardized response criteria specific to that disease orcondition. Tumor response can be assessed for changes in tumormorphology (i.e., overall tumor burden, tumor size, and the like) usingscreening techniques such as magnetic resonance imaging (MRI) scan,x-radiographic imaging, computed tomographic (CT) scan, bone scanimaging, endoscopy, and tumor biopsy sampling including bone marrowaspiration (BMA) and counting of tumor cells in the circulation.

In addition to these positive therapeutic responses, the subjectundergoing therapy may experience the beneficial effect of animprovement in the symptoms associated with the disease.

Treatment according to the present invention includes a “therapeuticallyeffective amount” of the medicaments used. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve a desired therapeutic result.

A therapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the medicaments to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects.

A “therapeutically effective amount” for tumor therapy may also bemeasured by its ability to stabilize the progression of disease. Theability of a compound to inhibit cancer may be evaluated in an animalmodel system predictive of efficacy in human tumors.

Alternatively, this property of a composition may be evaluated byexamining the ability of the compound to inhibit cell growth or toinduce apoptosis by in vitro assays known to the skilled practitioner. Atherapeutically effective amount of a therapeutic compound may decreasetumor size, or otherwise ameliorate symptoms in a subject. One ofordinary skill in the art would be able to determine such amounts basedon such factors as the subject's size, the severity of the subject'ssymptoms, and the particular composition or route of administrationselected.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. Parenteral compositions may beformulated in dosage unit form for ease of administration and uniformityof dosage. Dosage unit form as used herein refers to physically discreteunits suited as unitary dosages for the subjects to be treated; eachunit contains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

The specification for the dosage unit forms of the present invention aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

The efficient dosages and the dosage regimens for the bispecificantibodies used in the present invention depend on the disease orcondition to be treated and may be determined by the persons skilled inthe art.

An exemplary, non-limiting range for a therapeutically effective amountof an bispecific antibody used in the present invention is about 0.1-100mg/kg.

All cited references are herein expressly incorporated by reference intheir entirety.

Whereas particular embodiments of the invention have been describedabove for purposes of illustration, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

EXAMPLES

Examples are provided below to illustrate the present invention. Theseexamples are not meant to constrain the present invention to anyparticular application or theory of operation. For all constant regionpositions discussed in the present invention, numbering is according tothe EU index as in Kabat (Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, 5th Ed., United States Public Health Service,National Institutes of Health, Bethesda, entirely incorporated byreference). Those skilled in the art of antibodies will appreciate thatthis convention consists of nonsequential numbering in specific regionsof an immunoglobulin sequence, enabling a normalized reference toconserved positions in immunoglobulin families. Accordingly, thepositions of any given immunoglobulin as defined by the EU index willnot necessarily correspond to its sequential sequence.

General and specific scientific techniques are outlined in USPublications 2015/0307629, 2014/0288275 and WO2014/145806, all of whichare expressly incorporated by reference in their entirety andparticularly for the techniques outlined therein. Examples 1 and 2 fromU.S. Ser. No. 62/416,087, filed on Nov. 1, 2016 are expresslyincorporated by reference in their entirety, including the correspondingfigures.

XI. Example 1: Anti-PD-1 ABDs

A. 1A: Illustrative Anti-PD-1 ABDs

Examples of antibodies which bind PD-1 were generated in bivalent IgG1format with E233P/L234V/L235A/G236del/S267K substitutions, illustrativesequences for which are depicted in FIGS. 13A-13E. DNA encoding thevariable regions was generated by gene synthesis and was inserted intothe mammalian expression vector pTT5 by the Gibson Assembly method.Heavy chain VH genes were inserted via Gibson Assembly into pTT5encoding the human IgG1 constant region with the substitutions mentionedabove. Light chains VL genes were inserted into pTT5 encoding the humanCκ constant region. DNA was transfected into HEK293E cells forexpression. Additional PD-1 ABDs (including those derived from the aboveantibodies) were formatted as Fabs and scFvs for use inIL-15/Rα×anti-PD-1 bifunctional proteins of the invention, illustrativesequences for which are depicted respectively in FIGS. 14A-14E and inthe sequence listing.

B. 1B: Generation of Anti-PD-1 Clone 1C11

1. 1B(a): Generation and Screening of Anti-PD-1 Hybridoma

To develop additional PD-1 targeting arms for IL-15/Rα×anti-PD-1bifunctional proteins of the invention, monoclonal antibodies were firstgenerated by hybridoma technology through ImmunoPrecise, through theirStandard Method and Rapid Prime Method. For the Standard Method,antigen(s) was injected into 3 BALB/c mice. 7-10 days before beingsacrificed for hybridoma generation, the immunized mice received anantigen boost. Antibody titre is evaluated by ELISA on the antigen andthe best responding mice are chosen for fusion. A final antigen boost isgiven 4 days prior to fusion. Lymphocytes from the mice are pooled,purified then fused with SP2/0 myeloma cells. Fused cells are grown onHAT selective Single-Step cloning media for 10-12 days at which pointthe hybridomas were ready for screening. For the Rapid Prime method,antigen(s) was injected into 3 BALB/c mice. After 19 days, lymphocytesfrom all the mice are pooled, purified then fused with SP2/0 myelomacells. Fused cells are grown on HAT selective Single-Step cloning mediafor 10-12 days at which point the hybridomas were ready for screening.Antigen(s) used were mouse Fc fusion of human PD-1 (huPD-1-mFc), mouseFc fusion of cyno PD-1 (cynoPD-1-mFc), His-tagged human PD-1(huPD-1-His), His-tagged cyno PD-1 (cynoPD-1-His) or mixtures thereof.

Anti-PD-1 hybridoma clones generated as described above were subject totwo rounds of screening using Octet, a BioLayer Interferometry(BLI)-based method. Experimental steps for Octet generally included thefollowing: Immobilization (capture of ligand or test article onto abiosensor); Association (dipping of ligand- or test article-coatedbiosensors into wells containing serial dilutions of the correspondingtest article or ligand); and Dissociation (returning of biosensors towell containing buffer) in order to determine the affinity of the testarticles. A reference well containing buffer alone was also included inthe method for background correction during data processing.

For the first round, anti-mouse Fc (AMC) biosensors were used to capturethe clones with dips into 500 nM of bivalent human and cyno PD-1-Fc-His.For the second round, clones identified in the first round that werepositive for both human and cyno PD-1 were captured onto AMC biosensorsand dipped into 500 nM monovalent human and cyno PD-1-His.

2. 1B(b): Characterization of Clone 1C11

One hybridoma clone identified in Example 1B(a) was clone 1C11. DNAencoding the VH and VL of hybridoma clone 1C11 were generated by genesynthesis and subcloned using standard molecular biology techniques intoexpression vector pTT5 containing human IgG1 constant region withE233P/L234V/L235A/G236del/S267K substitutions to generate XENP21575,sequences for which are depicted in FIG. 15 .

a. 1B(b)(i): PD-L1 Blocking with Clone 1C11

Blocking of checkpoint receptor/ligand interaction is necessary for Tcell activation. The blocking ability of XENP21575 was investigated in acell binding assay. HEK293T cells transfected to express PD-1 wereincubated with XENP21575, as well as control antibodies. Followingincubation, a murine Fc fusion of PD-L1 was added and allowed toincubate. Binding of PD-L1-mFc to HEK293T cells was detected with ananti-murine IgG secondary antibody, data for which are depicted in FIG.16 .

b. 1B(b)(ii): T Cell Surface Binding of Clone 1C11

Binding of anti-PD-1 clone 1C11 to T cells was measured in anSEB-stimulated PBMC assay. Staphylococcal Enterotoxin B (SEB) is asuperantigen that causes T cell activation and proliferation in a mannersimilar to that achieved by activation via the T cell receptor (TCR),including expression of checkpoint receptors such as PD-1. Human PBMCswere stimulated with 100 ng/mL for 3 days. Following stimulation, PBMCswere incubated with the indicated test articles at indicatedconcentrations at 4° C. for 30 min. PBMCs were stained withanti-CD3-FITC (UCHT1) and APC labeled antibody for human immunoglobulinκ light chain. The binding of the test articles to T cells as indicatedby APC MFI on FITC+ cells is depicted in FIG. 17 .

c. 1B(b)(iii): T Cell Activation by Clone 1C11

T cell activation by clone 1C11, as indicated by cytokine secretion, wasinvestigated in an SEB-stimulated PBMC assay. Human PBMCs werestimulated with 500 ng/mL SEB for 2 days. Cells were then washed twicein culture medium and stimulated with 500 ng/mL SEB in combination withindicated amounts of indicated test articles for 24 hours. Supernatantswere then assayed for IL-2 and IFNγ by cells, data for which aredepicted in FIG. 18 .

3. 1B(c): Humanization of Clone 1C11

Clone 1C11 humanized using string content optimization (see, e.g., U.S.Pat. No. 7,657,380, issued Feb. 2, 2010). DNA encoding the heavy andlight chains were generated by gene synthesis and subcloned usingstandard molecular biology techniques into the expression vector pTT5.Sequences for an illustrative humanized variant of clone 1C11 inbivalent antibody format are depicted in depicted in FIG. 19 . Sequencesfor additional humanized variants of clone 1C11 are listed as XENPs22543, 22544, 22545, 22546, 22547, 22548, 22549, 22550, 22551, 22552,and 22554 in the figures and the sequence listing.

The affinity of XENP22553 was determined using Octet as generallydescribed in Example 1B(a). In particular, anti-human Fc (AHC)biosensors were used to capture the test article with dips into multipleconcentrations of histidine-tagged PD-1. The affinity result andcorresponding sensorgram are depicted in FIG. 20 .

XII. Example 2: IL-15/Rα-Fc

A. 2A: Engineering IL-15 Rα-Fc Fusion Proteins

In order to address the short half-life of IL-15/IL-15Rα heterodimers,we generated the IL-15/IL-15Rα(sushi) complex as a Fc fusion (hereonreferred to as IL-15/Rα-Fc fusion proteins) with the goal offacilitating production and promoting FcRn-mediated recycling of thecomplex and prolonging half-life.

Plasmids coding for IL-15 or IL-15Rα sushi domain were constructed bystandard gene synthesis, followed by subcloning into a pTT5 expressionvector containing Fc fusion partners (e.g., constant regions as depictedin FIG. 10A-10D). Cartoon schematics of illustrative IL-15/Rα-Fc fusionprotein formats are depicted in FIGS. 21A-21G.

Illustrative proteins of the IL-15/Rα-heteroFc format (FIG. 21A) includeXENP20818 and XENP21475, sequences for which are depicted in FIG. 22 ,with sequences for additional proteins of this format are XENPs 20819,21471, 21472, 21473, 21474, 21476, 21477 depicted in the figures. Anillustrative proteins of the scIL-15/Rα-Fc format (FIG. 21B) isXENP21478, sequences for which are depicted in FIG. 23 , with sequencesfor additional proteins of this format depicted as XENPs 21993, 21994,21995, 23174, 23175, 24477, 24480 in the figures. Illustrative proteinsof the ncIL-15/Rα-Fc format (FIG. 21C) include XENP21479, XENP22366, andXENP24348 sequences for which are depicted in FIG. 24 . An illustrativeprotein of the bivalent ncIL-15/Rα-Fc format (FIG. 21D) is XENP21978,sequences for which are depicted in FIG. 25 , with sequences foradditional proteins of this format depicted as XENP21979. Sequences foran illustrative protein of the bivalent scIL-15/Rα-Fc format (FIG. 21E)are depicted in FIG. 26 . An illustrative protein of the Fc-ncIL-15/Rαformat (FIG. 21F) is XENP22637, sequences for which are depicted in FIG.27 , with sequences for additional proteins of this format are depictedas XENP22638 in the figures. Sequences for an illustrative protein ofthe Fc-scIL-15/Rα format (FIG. 21G) are depicted in FIG. 28 .

Proteins were produced by transient transfection in HEK293E cells andwere purified by a two-step purification process comprising protein Achromatography (GE Healthcare) and anion exchange chromatography(HiTrapQ 5 mL column with a 5-40% gradient of 50 mM Tris pH 8.5 and 50mM Tris pH 8.5 with 1 M NaCl).

IL-15/Rα-Fc fusion proteins in the various formats as described abovewere tested in a cell proliferation assay. Human PBMCs were treated withthe test articles at the indicated concentrations. 4 days aftertreatment, the PBMCs were stained with anti-CD8-FITC (RPA-T8),anti-CD4-PerCP/Cy5.5 (OKT4), anti-CD27-PE (M-T271), anti-CD56-BV421(5.1H11), anti-CD16-BV421 (3G8), and anti-CD45RA-BV605 (Hi100) to gatefor the following cell types: CD4+ T cells, CD8+ T cells, and NK cells(CD56+/CD16+). Ki67 is a protein strictly associated with cellproliferation, and staining for intracellular Ki67 was performed usinganti-Ki67-APC (Ki-67) and Foxp3/Transcription Factor Staining Buffer Set(Thermo Fisher Scientific, Waltham, Mass.). The percentage of Ki67 onthe above cell types was measured using FACS (depicted in FIGS. 29A-29Cand FIGS. 30A-30C). The various IL-15/Rα-Fc fusion proteins inducedstrong proliferation of CD8+ T cells and NK cells. Notably, differencesin proliferative activity were dependent on the linker length on theIL-15-Fc side. In particular, constructs having no linker (hinge only),including XENP21471, XENP21474, and XENP21475, demonstrated weakerproliferative activity.

B. 2B: IL-15/Rα-Fc Fusion Proteins with Engineered Disulfide Bonds

To further improve stability and prolong the half-life of IL-15/Rα-Fcfusion proteins, we engineered disulfide bonds into the IL-15/Rαinterface. By examining the crystal structure of the IL-15/Rα complex,as well as by modeling using Molecular Operating Environment (MOE;Chemical Computing Group, Montreal, Quebec, Canada) software, wepredicted residues at the IL-15/Rα interface that may be substitutedwith cysteine in order to form covalent disulfide bonds, as depicted inFIG. 31 . Additionally, up to three amino acids following the sushidomain in IL-15Rα were added to the C-terminus of IL-15Rα(sushi) as ascaffold for engineering cysteines (illustrative sequences for which aredepicted in FIG. 32 ). Sequences for illustrative IL-15 andIL-15Rα(sushi) variants engineered with cysteines are respectivelydepicted in FIGS. 33 and 34

Plasmids coding for IL-15 or IL-15Rα(sushi) were constructed by standardgene synthesis, followed by subcloning into a pTT5 expression vectorcontaining Fc fusion partners (e.g., constant regions as depicted inFIGS. 10A-10D. Residues identified as described above were substitutedwith cysteines by standard mutagenesis techniques. Cartoon schematics ofIL-15/Rα-Fc fusion proteins with engineered disulfide bonds are depictedin FIGS. 35A-35D.

Illustrative proteins of the dsIL-15/Rα-heteroFc format (FIG. 35A)include XENP22013, XENP22014, XENP22015, and XENP22017, sequences forwhich are depicted in FIG. 36 . Illustrative proteins of thedsIL-15/Rα-Fc format (FIG. 35B) include XENP22357, XENP22358, XENP22359,XENP22684, and XENP22361, sequences for which are depicted in FIG. 37 ,with sequences for additional proteins of this format depicted as XENPs22360, 22362, 22363, 22364, 22365, 22366 in the figures. Illustrativeprotein of the bivalent dsIL-15/Rα-Fc format (FIG. 35C) includeXENP22634, XENP22635, and XENP22636, sequences for which are depicted inFIG. 38 , with sequences for additional proteins of this format depictedas XENP22687 in the figures. Illustrative proteins of the Fc-dsIL-15/Rαformat (FIG. 35D) include XENP22639 and XENP22640, sequences for whichare depicted in FIG. 39 .

Proteins were produced by transient transfection in HEK293E cells andwere purified by a two-step purification process comprising protein Achromatography (GE Healthcare) and anion exchange chromatography(HiTrapQ 5 mL column with a 5-40% gradient of 50 mM Tris pH 8.5 and 50mM Tris pH 8.5 with 1 M NaCl).

After the proteins were purified, they were characterized by capillaryisoelectric focusing (CEF) for purity and homogeneity. CEF was performedusing LabChip GXII Touch HT (PerkinElmer, Waltham, Mass.) using ProteinExpress Assay LabChip and Protein Express Assay Reagent Kit carried outusing the manufacturer's instructions. Samples were run in duplicate,one under reducing (with dithiothreitol) and the other undernon-reducing conditions. Many of the disulfide bonds were correctlyformed as indicated by denaturing non-reducing CEF, where the largermolecular weight of the covalent complex can be seen when compared tothe controls without engineered disulfide bonds (FIG. 40 ).

The proteins were then tested in a cell proliferation assay. IL-15/Rα-Fcfusion proteins (with or without engineered disulfide bonds) or controlswere incubated with PBMCs for 4 days. Following incubation, PBMCs werestained with anti-CD4-PerCP/Cy5.5 (RPA-T4), anti-CD8-FITC (RPA-T8),anti-CD45RA-BV510 (HI100), anti-CD16-BV421 (3G8), anti-CD56-BV421(HCD56), anti-CD27-PE (0323), and anti-Ki67-APC (Ki-67) to mark variouscell populations and analyzed by FACS as generally described in Example2A. Proliferation of NK cells, CD4+ T cells, and CD8+ T cells asindicated by Ki67 expression are depicted in FIGS. 41A-41C. Each of theIL-15/Rα-Fc fusion proteins and the IL-15 control induced strongproliferation of NK cells, CD8+ T cells, and CD4+ T cells.

C. 2C:IL-15/Rα-Fc Fusion Proteins Engineered for Lower Potency andIncreased PK and Half-Life

In order to further improve PK and prolong half-life, we reasoned thatdecreasing the potency of IL-15 would decrease the antigen sink, andthus, increase the half-life. By examining the crystal structure of theIL-15:IL-2Rß and IL-15:common gamma chain interfaces, as well as bymodeling using MOE software, we predicted residues at these interfacesthat may be substituted in order to reduce potency. FIG. 42 depicts astructural model of the IL-15:receptor complexes showing locations ofthe predicted residues where we engineered isosteric substitutions (inorder to reduce the risk of immunogenicity). Sequences for illustrativeIL-15 variants engineered for reduced potency are depicted in FIG. 43 .

Plasmids coding for IL-15 or IL-15Rα(sushi) were constructed by standardgene synthesis, followed by subcloning into a pTT5 expression vectorcontaining Fc fusion partners (e.g., constant regions as depicted inFIG. 10 a -10D). Substitutions identified as described above wereincorporated by standard mutagenesis techniques. Sequences forillustrative IL-15/Rα-Fc fusion proteins of the “IL-15/Rα-heteroFc”format engineered for reduced potency are depicted in FIG. 44 , withadditional sequences depicted as XENPs 22815, 22816, 22817, 22818,22819, 22820, 22823, 22824, 22825, 22826, 22827, 22828, 22829, 22830,22831, 22832, 22833, 22834, 23555, 23559, 23560, 24017, 24020, and24043, and 24048 in the figures and the sequence listing. Sequences forillustrative IL-15/Rα-Fc fusion proteins of the “scIL-15/Rα-Fc” formatengineered for reduced potency are depicted in FIG. 45 , with additionalsequences depicted as XENPs 24013, 24014, and 24016 in the figures.Sequences for illustrative IL-15/Rα-Fc fusion proteins of the“ncIL-15/Rα-Fc” format engineered for reduced potency are depicted inFIG. 46 . Sequences for illustrative ncIL-15/Rα heterodimers engineeredfor reduced potency are depicted in FIG. 47 , with additional sequencesdepicted as XENPs 22791, 22792, 22793, 22794, 22795, 22796, 22803,22804, 22805, 22806, 22807, 22808, 22809, 22810, 22811, 22812, 22813,and 22814 in the figures. Sequences for an illustrative IL-15/Rα-Fcfusion protein of the “bivalent ncIL-15/Rα-Fc” format engineered forreduced potency are depicted in FIG. 48 . Sequences for illustrativeIL-15/Rα-Fc fusion proteins of the “dsIL-15/Rα-Fc” format engineered forreduced potency are depicted in FIG. 49 . Proteins were produced bytransient transfection in HEK293E cells and were purified by a two-steppurification process comprising protein A chromatography (GE Healthcare)and anion exchange chromatography (HiTrapQ 5 mL column with a 5-40%gradient of 50 mM Tris pH 8.5 and 50 mM Tris pH 8.5 with 1 M NaCl).

1. 2C(a): In Vitro Activity of Variant IL-15/Rα-Fc Fusion ProteinsEngineered for Decreased Potency

The variant IL-15/Rα-Fc fusion proteins were tested in a number of cellproliferation assays.

In a first cell proliferation assay, IL-15/Rα-Fc fusion proteins (withor without engineered substitutions) or control were incubated withPBMCs for 4 days. Following incubation, PBMCs were stained withanti-CD4-Evolve605 (SK-3), anti-CD8-PerCP/Cy5.5 (RPA-T8),anti-CD45RA-APC/Cy7 (HI100), anti-CD16-eFluor450 (CB16),anti-CD56-eFluor450 (TULY56), anti-CD3-FITC (OKT3), and anti-Ki67-APC(Ki-67) to mark various cell populations and analyzed by FACS asgenerally described in Example 2A. Proliferation of NK cells, CD8+ Tcells, and CD4+ T cells as indicated by Ki67 expression are depicted inFIGS. 50A-50C and 51 . Most of the IL-15/Rα-Fc fusion proteins inducedproliferation of each cell population; however, activity varieddepending on the particular engineered substitutions.

In a second cell proliferation assay, IL-15/Rα-Fc fusion proteins (withor without engineered substitutions) were incubated with PBMCs for 3days. Following incubation, PBMCs were stained with anti-CD3-FITC(OKT3), anti-CD4-Evolve604 (SK-3), anti-CD8-PerCP/Cy5.5 (RPA-T8),anti-CD16-eFluor450 (CB16), anti-CD56-eFluor450 (TULY56), anti-CD27-PE(0323), anti-CD45RA-APC/Cy7 (HI100) and anti-Ki67-APC (20Raj1)antibodies to mark various cell populations. FIGS. 52A-52C and 53A-53Cdepict selection of various cell populations following incubation withXENP22821 by FACS. Lymphocytes were first gated on the basis of sidescatter (SSC) and forward scatter (FSC) (FIG. 52A). Lymphocytes werethen gated based on CD3 expression (FIG. 52B). Cells negative for CD3expression were further gated based on CD16 expression to identify NKcells (CD16+) (FIG. 52C). CD3+ T cells were further gated based on CD4and CD8 expression to identify CD4+ T cells, CD8+ T cells, and γδ Tcells (CD3+CD4−CD8−) (FIG. 53A). The CD4+ and CD8+ T cells were gatedfor CD45RA expression as shown respectively in FIGS. 53B-53C. Finally,the proliferation of the various cell populations were determined basedon percentage Ki67 expression, and the data are shown in FIGS. 55A-55D.NK and CD8+ T cells are more sensitive than CD4+ T cells to IL-15/Rα-Fcfusion proteins, and as above, proliferative activity varied dependingon the particular engineered substitutions. FIG. 55D shows the foldchange in EC50 of various IL-15/Rα-Fc fusion proteins relative tocontrol XENP20818. FIGS. 54A and 54B further depict the activation oflymphocytes following treatment with IL-15/Rα-Fc fusion proteins bygating for the expression of CD69 and CD25 (T cell activation markers)before and after incubation of PBMCs with XENP22821.

In a third experiment, additional variant IL-15/Rα-Fc fusion proteinswere incubated with human PBMCs for 3 days at 37° C. Followingincubation, PBMCs were stained with anti-CD3-FITC (OKT3), anti-CD4-SB600(SK-3), anti-CD8-PerCP/Cy5.5 (RPA-T8), anti-CD45RA-APC/Cy7 (HI100),anti-CD16-eFluor450 (CB16), anti-CD25-PE (M-A251), and anti-Ki67-APC(Ki-67) to mark various cell populations and analyzed by FACS asgenerally described in Example 2A. Proliferation of CD8+ (CD45RA−) Tcells, CD4+ (CD45RA−) T cells, γδ T cells, and NK cells as indicated byKi67 expression are depicted in FIGS. 56A-D.

In a fourth experiment, human PBMCs were incubated with the additionalIL-15/Rα-Fc variants at the indicated concentrations for 3 days.Following incubation, PBMCs were stained with anti-CD3-FITC (OKT3),anti-CD4 (SB600), anti-CD8-PerCP/Cy5.5 (RPA-T8), anti-CD16-eFluor450(CB16), anti-CD25-PE (M-A251), anti-CD45RA-APC/Cy7 (HI100), andanti-Ki67-APC (Ki67) and analyzed by FACS as generally described inExample 2A. Percentage of Ki67 on CD8+ T cells, CD4+ T cells and NKcells following treatment are depicted in FIG. 57 .

In a fifth experiment, variant IL-15/Rα-Fc fusion proteins wereincubated with human PBMCs for 3 days at 37° C. Following incubation,cells were stained with anti-CD3-PE (OKT3), anti-CD4-FITC (RPA-T4),anti-CD8α-BV510 (SK1), anti-CD8ß-APC (2ST8.5H7), anti-CD16-BV421 (3G8),anti-CD25-PerCP/Cy5.5 (M-A251), anti-CD45RA-APC/Cy7 (HI100),anti-CD56-BV605 (NCAM16.2), and anti-Ki67-PE/Cy7 (Ki-67) and analyzed byFACS as generally described in Example 2A. Percentage of Ki67 on CD8+ Tcells, CD4+ T cells, γδ T cells, and NK cells are depicted in FIGS.58A-58E.

In a sixth experiment, variant IL-15/Rα-Fc fusion proteins wereincubated with human PBMCs for 3 days at 37° C. Following incubation,cells were stained with anti-CD3-PE (OKT3), anti-CD4-FITC (RPA-T4),anti-CD8α-BV510 (SK1), anti-CD8ß-APC (SIDI8BEE), anti-CD16-BV421 (3G8),anti-CD25-PerCP/Cy5.5 (M-A251), anti-CD45RA-APC/Cy7 (HI100),anti-CD56-BV605 (NCAM16.2), and anti-Ki67-PE/Cy7 (Ki-67) and analyzed byFACS as generally described in Example 2A. Percentage of Ki67 on CD8+ Tcells, CD4+ T cells, γδ T cells, and NK cells are depicted in FIGS.59A-59E.

In a seventh experiment, variant IL-15/Rα-Fc fusion proteins wereincubated with human PBMCs at the indicated concentrations for 3 days at37° C. Following incubation, PBMCs were stained with anti-CD3-PE (OKT3),anti-CD4-FITC (RPA-T4), anti-CD8-APC (RPA-T8), anti-CD16-BV605 (3G8),anti-CD25-PerCP/Cy5.5 (M-A251), anti-CD45RA-APC/Fire750 (HI100) andanti-Ki67-PE/Cy7 (Ki-67) and analyzed by FACS as generally described inExample 2A. Percentage Ki67 on CD8+ T cells, CD4+ T cells, γδ T cellsand NK (CD16+) cells are depicted in FIGS. 60A-60D. The data show thatthe ncIL-15/Rα-Fc fusion protein XENP21479 is the most potent inducer ofCD8+ T cell, CD4+ T cell, NK (CD16+) cell, and γδ T cell proliferation.Each of the scIL-15/Rα-Fc fusion proteins were less potent thanXENP21479 in inducing proliferation, but differences were dependent onboth the linker length, as well as the particular engineeredsubstitutions.

In an eighth experiment, variant IL-15/Rα-Fc fusion proteins wereincubated with human PBMCs at the indicated concentrations for 3 days at37° C. Following incubation, PBMCs were stained with anti-CD3-PE (OKT3),anti-CD4-FITC (RPA-T4), anti-CD8-APC (RPA-T8), anti-CD16-BV605 (3G8),anti-CD25-PerCP/Cy5.5 (M-A251), anti-CD45RA-APC/Fire750 (HI100) andanti-Ki67-PE/Cy7 (Ki-67) and analyzed by FACS as generally described inExample 2A. Percentage Ki67 on CD8+ T cells, CD4+ T cells, γδ T cellsand NK (CD16+) cells are respectively depicted in FIGS. 61A-61D. Asabove, the data show that the ncIL-15/Rα-Fc fusion protein XENP21479 isthe most potent inducer of CD8+ T cell, CD4+ T cell, NK (CD16+) cell,and γδ T cell proliferation. Notably, introduction of Q108E substitutioninto the ncIL-15/Rα-Fc format (XENP24349) drastically reduces itsproliferative activity in comparison to wildtype (XENP21479).

2. 2C(b): PK of IL-15/Rα-Fc Fusion Proteins Engineered for ReducedPotency

In order to investigate if IL-15/Rα-Fc fusion proteins engineered forreduced potency had improved half-life and PK, we examined thesevariants in a PK study in C57BL/6 mice. Two cohorts of mice (5 mice pertest article per cohort) were dosed with 0.1 mg/kg of the indicated testarticles via IV-TV on Day 0. Serum was collected 60 minutes after dosingand then on Days 2, 4, and 7 for Cohort 1 and Days 1, 3, and 8 forCohort 2. Serum levels of IL-15/Rα-Fc fusion proteins were determinedusing anti-IL-15 and anti-IL-15Rα antibodies in a sandwich ELISA. Theresults are depicted in FIG. 62 . FIG. 63 depicts the correlationbetween potency and half-life of the test articles. As predicted,variants with reduced potency demonstrated substantially longerhalf-life. Notably, half-life was improved up to almost 9 days (seeXENP22821 and XENP22822), as compared to 0.5 days for the wild-typecontrol XENP20818.

XIII. Example 3: IL-15/Rα×Anti-PD-1 Bifunctionals

A. 3A: Generation and Physical Characterization of IL-15/Rα×Anti-PD-1Bifunctionals

Plasmids coding for IL-15, IL-15Rα sushi domain, or the anti-PD-1variable regions were constructed by standard gene synthesis, followedby subcloning into a pTT5 expression vector containing Fc fusionpartners (e.g., constant regions as depicted in FIG. 11. Cartoonschematics of illustrative IL-15/Rα×anti-PD-1 bifunctionals are depictedin FIG. 64 .

The “scIL-15/Rα×scFv” format (FIG. 64A) comprises IL-15Rα(sushi) fusedto IL-15 by a variable length linker (termed “scIL-15/Rα”) which is thenfused to the N-terminus of a heterodimeric Fc-region, with an scFv fusedto the other side of the heterodimeric Fc. Sequences for illustrativebifunctional proteins of this format are depicted in FIG. 65 .

The “scFv×ncIL-15/Rα” format (FIG. 64B) comprises an scFv fused to theN-terminus of a heterodimeric Fc-region, with IL-15Rα(sushi) fused tothe other side of the heterodimeric Fc, while IL-15 is transfectedseparately so that a non-covalent IL-15/Rα complex is formed. Sequencesfor illustrative bifunctional proteins of this format are depicted inFIG. 66 .

The “scFv×dsIL-15/Rα” format (FIG. 64C) is the same as the“scFv×ncIL-15/Rα” format, but wherein IL-15Rα(sushi) and IL-15 arecovalently linked as a result of engineered cysteines. Sequences forillustrative bifunctional proteins of this format are depicted in FIG.67 .

The “scIL-15/Rα×Fab” format (FIG. 64D) comprises IL-15Rα(sushi) fused toIL-15 by a variable length linker (termed “scIL-15/Rα”) which is thenfused to the N-terminus of a heterodimeric Fc-region, with a variableheavy chain (VH) fused to the other side of the heterodimeric Fc, whilea corresponding light chain is transfected separately so as to form aFab with the VH. Sequences for illustrative bifunctional proteins ofthis format are depicted in FIG. 68 .

The “ncIL-15/Rα×Fab” format (FIG. 64E) comprises a VH fused to theN-terminus of a heterodimeric Fc-region, with IL-15Rα(sushi) fused tothe other side of the heterodimeric Fc, while a corresponding lightchain is transfected separately so as to form a Fab with the VH, andwhile IL-15 is transfected separately so that a non-covalent IL-15/Rαcomplex is formed. Sequences for illustrative bifunctional proteins ofthis format are depicted in FIG. 69 .

The “dsIL-15/Rα×Fab” format (FIG. 64F) is the same as the“ncIL-15/Rα×Fab” format, but wherein IL-15Rα(sushi) and IL-15 arecovalently linked as a result of engineered cysteines. Sequences forillustrative bifunctional proteins of this format are depicted in FIG.70 .

The “mAb-scIL-15/Rα” format (FIG. 64G) comprises VH fused to theN-terminus of a first and a second heterodimeric Fc, with IL-15 is fusedto IL-15Rα(sushi) which is then further fused to the C-terminus of oneof the heterodimeric Fc-region, while corresponding light chains aretransfected separately so as to form a Fabs with the VHs. Sequences forillustrative bifunctional proteins of this format are depicted in FIG.71 .

The “mAb-ncIL-15/Rα” format (FIG. 64H) comprises VH fused to theN-terminus of a first and a second heterodimeric Fc, with IL-15Rα(sushi)fused to the C-terminus of one of the heterodimeric Fc-region, whilecorresponding light chains are transfected separately so as to form aFabs with the VHs, and while and while IL-15 is transfected separatelyso that a non-covalent IL-15/Rα complex is formed. Sequences forillustrative bifunctional proteins of this format are depicted in FIG.72 .

The “mAb-dsIL-15/Rα” format (FIG. 64I) is the same as the“mAb-ncIL-15/Rα” format, but wherein IL-15Rα(sushi) and IL-15 arecovalently linked as a result of engineered cysteines. Sequences forillustrative bifunctional proteins of this format are depicted in FIG.73 .

The “central-IL-15/Rα” format (FIG. 64J) comprises a VH recombinantlyfused to the N-terminus of IL-15 which is then further fused to one sideof a heterodimeric Fc and a VH recombinantly fused to the N-terminus ofIL-15Rα(sushi) which is then further fused to the other side of theheterodimeric Fc, while corresponding light chains are transfectedseparately so as to form a Fabs with the VHs. Sequences for illustrativebifunctional proteins of this format are depicted in FIG. 74 .

The “central-scIL-15/Rα” format (FIG. 64K) comprises a VH fused to theN-terminus of IL-15Rα(sushi) which is fused to IL-15 which is thenfurther fused to one side of a heterodimeric Fc and a VH fused to theother side of the heterodimeric Fc, while corresponding light chains aretransfected separately so as to form a Fabs with the VHs. Sequences forillustrative bifunctional proteins of this format are depicted in FIG.75 .

IL-15/Rα×anti-PD-1 bifunctional proteins were characterized bysize-exclusion chromatography (SEC) and capillary isoelectric focusing(CEF) for purity and homogeneity.

The proteins were analyzed using SEC to measure their size (i.e.hydrodynamic volume) and determine the native-like behavior of thepurified samples. The analysis was performed on an Agilent 1200high-performance liquid chromatography (HPLC) system. Samples wereinjected onto a Superdex™ 200 10/300 GL column (GE Healthcare LifeSciences) at 1.0 mL/min using 1×PBS, pH 7.4 as the mobile phase at 4° C.for 25 minutes with UV detection wavelength at 280 nM. Analysis wasperformed using Agilent OpenLab Chromatography Data System (CDS)ChemStation Edition AIC version C.01.07. Chromatogram for anillustrative IL-15/Rα×anti-PD-1 bifunctional XENP21480 in theIL-15/Rα×scFv format is shown in FIG. 76B.

The proteins were analyzed electrophoretically via CEF using LabChipGXII Touch HT (PerkinElmer, Waltham, Mass.) using Protein Express AssayLabChip and Protein Express Assay Reagent Kit carried out using themanufacturer's instructions. Samples were run in duplicate, one underreducing (with dithiothreitol) and the other under non-reducingconditions. Gel image for XENP21480 is shown in FIG. 76C.

Affinity screens of the bifunctional proteins for IL-2RB and PD-1 wereperformed using Octet as generally described in Example 1B(a). In afirst screen, anti-human Fc (AHC) biosensors were used to capture thetest articles and then dipped into multiple concentration of IL-2RB (R&DSystems, Minneapolis, Minn.) or histidine-tagged PD-1 for KDdetermination. The affinity result and corresponding sensorgrams forXENP21480 are depicted in FIGS. 76D-76E. In a second screen, a HIS 1Kbiosensors were used to capture either histidine-tagged IL-2RB:commongamma chain complex-Fc fusion or histidine-tagged PD-1-Fc fusion andthen dipped into 2 different batches of XENP25850, sensorgrams for whichare depicted in FIG. 77 .

Stability of the bifunctional proteins were evaluated using DifferentialScanning Fluorimetry (DSF). DSF experiments were performed using aBio-Rad CFX Connect Real-Time PCR Detection System. Proteins were mixedwith SYPRO Orange fluorescent dye and diluted to 0.2 mg/mL in PBS. Thefinal concentration of SYPRO Orange was 10×. After an initial 10 minuteincubation period at 25° C., proteins were heated from 25 to 95° C.using a heating rate of 1° C./min. A fluorescence measurement was takenevery 30 sec. Melting temperatures (Tm) were calculated using theinstrument software. The stability result and corresponding meltingcurve for XENP21480 are depicted in FIG. 76F.

B. 3B: Activity of IL-15/Rα×Anti-PD-1 Bifunctionals in CellProliferation Assays

An illustrative IL-15/Rα×anti-PD-1 bifunctional protein XENP21480 andcontrols were tested in a cell proliferation assay. Human PBMCs weretreated with the test articles at the indicated concentrations. 4 daysafter treatment, the PBMCs were stained with anti-CD8-FITC (RPA-T8),anti-CD4-PerCP/Cy5.5 (OKT4), anti-CD27-PE (M-T271), anti-CD56-BV421(5.1H11), anti-CD16-BV421 (3G8), and anti-CD45RA-BV605 (Hi100) to gatefor the following cell types: CD4+ T cells, CD8+ T cells, and NK cells(CD56+/CD16+). Ki67 is a protein strictly associated with cellsproliferation, and staining for intracellular Ki67 was performed usinganti-Ki67-APC (Ki-67) and Foxp3/Transcription Factor Staining Buffer Set(Thermo Fisher Scientific, Waltham, Mass.). The percentage of Ki67 onthe above cell types was measured using FACS (depicted in FIGS.78A-78C).

C. 3C: Activity of IL-15/Rα×Anti-PD-1 Bifunctionals in an SEB-StimulatedPBMC Assay

Human PBMCs from multiple donors were stimulated with 10 ng/mL of SEBfor 72 hours in combination with 20 μg/mL of an illustrativeIL-15/Rα×anti-PD-1 bifunctional protein or controls. After treatment,supernatant was collected and assayed for IL-2, data for which isdepicted in FIG. 79 .

D. 3D: IL-15/Rα×Anti-PD-1 Bifunctionals Enhance Engraftment and DiseaseActivity in Human PBMC-Engrafted NSG Mice

An illustrative IL-15/Rα×anti-PD-1 was evaluated in a Graft-versus-HostDisease (GVHD) model conducted in NSG (NOD-SCID-gamma) immunodeficientmice. When the NSG mice are injected with human PBMCs, the human PBMCsdevelop an autoimmune response against mouse cells. Treatment of NSGmice injected with human PBMCs followed with IL-15/Rα×anti-PD-1de-repress and proliferate the engrafted T cells and enhancesengraftment.

10 million human PBMCs were engrafted into NSG mice via IV-OSP on Day −8followed by dosing with the indicated test articles at the indicatedconcentrations on Day 0. IFNγ levels and human CD45+ lymphocytes, CD8+ Tcell and CD4+ T cell counts were measured at Days 4, 7, and 11. FIG. 80depicts IFNγ levels in mice serum on Days 4, 7, and 11. FIGS. 81A-81Crespectively depict CD8+ T cell counts on Days 4, 7, and 11. FIGS.82A-82C respectively depict CD4+ T cell counts on Days 4, 7, and 11.FIGS. 83A-83C respectively depict CD45+ cell counts on Days 4, 7, and11. Body weight of the mice were also measured on Days 4, 7, and 11 anddepicted as percentage of initial body weight in FIG. 84 .

What is claimed is:
 1. A bispecific heterodimeric protein comprising: a)a fusion protein comprising a first protein domain, a second proteindomain, and a first Fc domain, wherein said first protein domain iscovalently attached to the N-terminus of said second protein domainusing a first domain linker, wherein said second protein domain iscovalently attached to the N-terminus of said first Fc domain using asecond domain linker, and wherein said first protein domain comprises ahuman IL-15Rα protein and said second protein domain comprises a variantof a human IL-15 protein of SEQ ID NO:2 comprising amino acidsubstitutions selected from the group consisting of (i) N4D/N65D, (ii)D30N/N65D, and (iii) D30N/E64Q/N65D; and b) an antibody fusion proteincomprising a PD-1 antigen binding domain (ABD) and a second Fc domain,wherein said PD-1 antigen binding domain is covalently attached to theN-terminus of said second Fc domain, and said PD-1 antigen bindingdomain is a single chain variable fragment (scFv) or a Fab fragment. 2.The bispecific heterodimeric protein of claim 1, wherein said variantIL-15 protein has a sequence selected from SEQ ID NO:374, SEQ ID NO:375and SEQ ID NO:380.
 3. The bispecific heterodimeric protein of claim 1,wherein said anti-PD-1 ABD is a Fab, and said bispecific heterodimericprotein further comprises a light chain.
 4. A nucleic acid compositioncomprising: a) a first nucleic acid encoding said fusion protein ofclaim 3; b) a second nucleic acid encoding said antibody fusion proteinof claim 3; and c) a third nucleic acid encoding said light chain ofclaim
 3. 5. An expression vector composition comprising: a) a firstexpression vector comprising said first nucleic acid of claim 4; b) asecond expression vector comprising said second nucleic acid of claim 4;and c) a third expression vector comprising said third nucleic acid ofclaim
 4. 6. A host cell comprising the expression vector composition ofclaim
 5. 7. A method of producing a bispecific heterodimeric proteincomprising culturing the host cell of claim 6 under conditions wherebysaid protein is expressed, and recovering said protein.
 8. A bispecificheterodimeric protein comprising: a) a fusion protein comprising a firstprotein domain and a first Fc domain, wherein said first protein domainis covalently attached to the N-terminus of said first Fc domain using adomain linker and said first protein domain comprises a human IL-15Rαprotein; b) a second protein domain noncovalently attached to said firstprotein domain, said second protein domain comprises a variant of humanIL-15 protein of SEQ ID NO:2 comprising amino acid substitutionsselected from the group consisting of (i) N4D/N65D, (ii) D30N/N65D, and(iii) D30N/E64Q/N65D; and c) an antibody fusion protein comprising anPD-1 antigen binding domain and a second Fe domain, wherein said PD-1antigen binding domain is covalently attached to the N-terminus of saidsecond Fc domain and said PD-I antigen binding domain is a single chainvariable fragment (scFv) or a Fab fragment.
 9. A bispecificheterodimeric protein comprising: a) a first antibody fusion proteincomprising a first PD-1 antigen binding domain and a first Fc domain,wherein said first PD-1 antigen binding domain is covalently attached tothe N-terminus of said first Fc domain via a first domain linker, andsaid first PD-1 antigen binding domain is a single chain variablefragment (scFv) or a Fab fragment; b) a second antibody fusion proteincomprising a second PD-1 antigen binding domain, a second Fc domain, anda first protein domain, wherein said second PD-1 antigen binding domainis covalently attached to the N-terminus of said second Fc domain via asecond domain linker, said first protein domain is covalently attachedto the C-terminus of said second Fc domain via a third domain linker,said second PD-1 antigen binding domain is a single chain variablefragment (scFv) or a Fab fragment, and said first protein domaincomprises a human IL-15Rα protein; and (c) a second protein domainnoncovalently attached to said first protein domain of said secondantibody fusion protein and comprising a variant of a human IL-15protein of SEQ ID NO:2 comprising amino acid substitutions selected fromthe group consisting of (i) N4D/N65D, (ii) D30N/N65D, and (iii)D30N/E64Q/N65D.